Additive channels

ABSTRACT

Compositions, devices and methods are described for preventing, reducing, controlling or delaying adhesion, adsorption, surface-mediated clot formation, or coagulation in a microfluidic device or chip. In one embodiment, blood (or other fluid with blood components) that contains anticoagulant is introduced into a microfluidic device comprising one or more additive channels containing one or more reagents that will re-activate the native coagulation cascade in the blood that makes contact with it “on-chip” before moving into the experimental region of the chip.

FIELD OF THE INVENTION

The present invention contemplates compositions, devices and methods ofpreventing, reducing, controlling or delaying adhesion, adsorption,swine-mediated clot formation, or coagulation in a microfluidic deviceor chip. In one embodiment, blood (or other fluid with blood components)that contains anticoagulant is introduced into a microfluidic devicecomprising one or more additive channels containing one or more reagentsthat will re-activate the native coagulation cascade in the blood thatmakes contact with it “on-chip” before moving into the experimentalregion of the chip.

BACKGROUND

Blood clotting, a process that relies on adhesion of platelets andproteins to a surface as a first step, can be a problem when blood isintroduced into a microfluidic device. Undesired clot formation can makemany desired blood tests impossible. Heparin coating of surfaces cancontrol blood clotting to a limited extent, See Barstad, R. M. et al.,Thrombosis and Haemostasis 79, 302-305 (1998). Certain polymericspecies, such as polyethylene glycol (PEG) chains, can influence thesurface hydration layer to prevent protein adsorption. See Chen, S. etal., Polymer 51, 5283-5293 (2010). However, they are not fully effectiveand soluble anticoagulants still must be added to the blood.

What is needed is better control over blood clotting in a microfluidicdevice.

SUMMARY OF THE INVENTION

The present invention contemplates compositions, devices and methods ofpreventing, reducing, controlling or delaying adhesion, adsorption,surface-mediated clot formation, or coagulation in a microfluidic deviceor chip. In one embodiment, blood (or other fluid with blood components)that contains anticoagulant is introduced into a microfluidic devicecomprising one or more additive channels containing one or more reagentsthat will reactivate the native coagulation cascade in the blood thatmakes contact with it “on-chip” before moving into the active orexperimental region of the chip.

In one embodiment, fixatives are contemplated as additives for theadditive channel (which can be useful for capturing the cells andplatelets in their state immediately after contact with the cells in thechip). In one embodiment, oil is contemplated as an additive for theadditive channel, to form blood-containing droplets (e.g. forsequestering blood samples from different time-points in the run, andanalyzing them separately afterwards), etc. The addition of an additivechannel near the outlet allows (in a versatile way) quick treatment ofblood samples as they leave the chip. Such treated blood samples arecontemplated to enable downstream analysis including but not limited tonew types of analysis from the use of the additive channel for treatingblood components as it leaves the chip.

Proposed mechanisms of hemostasis, platelet activation, and aggregationunder arterial flow show that the dynamical cross-talk between theendothelium and platelets (as well as other cells such as leukocytes,microparticles, etc.) may cause blood cells to tether, detach, andtranslocate in space and time in vivo. Kulkarni, S. et al. “A revisedmodel of platelet aggregation.” J. Clinical Investigation 105(6),783-791 (2000). Indeed, part of the reason why it has been difficult toassess platelet function accurately and reliably in vitro could be dueto the fact that the existing tests do not incorporate a relevant shearstress environment or assess the contribution of endothelial function.Jackson, S. P. “The growing complexity of platelet aggregation.” Blood109(12), 5087-5095 (2007).

Microfluidic devices (or “chips”) containing living cells recreate thephysiological tissue-tissue interfaces and permit fluid flow. See U.S.Pat. No. 8,647,861, hereby incorporated by reference. Such devicessubject the cells to shear stress. In contrast to static 2D culture,microchannels allow the perfusion of cell culture medium throughout thecell culture during in vitro studies and as such offer a more invivo-like physical environment. In simple terms, an inlet port allowsinjection of fluids such as blood, serum, plasma, cell culture medium(and the like) into a microfluidic channel or chamber (with or withoutcells). In one embodiment, the present invention contemplates acell-laden microfluidic channel or chamber. An outlet port then permitsthe exit of remaining fluid as well as harmful metabolic by-products.Thus, microfluidic devices may be more reliable in vitro testingplatforms for platelet analysis, including clot formation.

In one embodiment, the microfluidic device or chip is perfused byinserting it into a perfusion manifold or “pod.” Perfusion manifolds ofthis type are described in U.S. patent application Ser. No. 15/248,509,hereby incorporated by reference.

While soluble anticoagulants prevent or at least reduce clot formationin a microfluidic, device, they make the control over clot formation(when it is desired) difficult. The formation of aggregates and clotsmay result in contamination or blockage of the microchannels. Oneapproach is to use off-chip mixing of blood with anticoagulants prior tocontacting the chip with the blood. However, when on-chip coagulation isdesired (or at least the possibility of coagulation is desired), thisrequires contact (and even mixing) with a reagent that re-activates thecoagulation cascade (e.g. calcium).

Treating all of the blood (i.e. treating in bulk) with a reagent thatre-activates the coagulation cascade prior to introducing the blood intothe microfluidic device or chip is problematic. Microfluidic deviceshave slow flow rates. By the time the majority of the blood has enteredthe microfluidic device, if treated in bulk, it is likely to havecoagulated. This would render the microchannel, if not the entiredevice, inoperable.

Another approach is to use on-chip contacting of blood (or other fluidwith blood components) with one or more reagents that re-activate thecoagulation cascade. Rather than treatment in bulk, only that fractionof the blood in contact with the reagent(s) that re-activate thecoagulation cascade can clot. If this is done as the blood enters theactive region, or immediately prior, clotting is only possible in theactive region. This provides control over clotting.

With this said, on-chip mixing is complicated by dispersion of reagentsalong the microchannel, slow or incomplete mixing, and surfaceadsorption (due to the high surface area-to-volume ratio in microfluidicdevices). To improve mixing, the present invention contemplatesmicrofluidic devices with one or more additive channels. In oneembodiment, blood (or other fluid with blood components) that containsanticoagulant is introduced into a microfluidic device (e.g. through aninput port) comprising one or more additive channels (e.g. positioned ator near the input port) containing one or more reagents that willre-activate the native coagulation cascade in the portion of the bloodthat makes contact with it “on-chip” before moving into the active orexperimental region of the chip.

While one additive channel can be used, it has been found empiricallythat two additive channels (one on either side of the input port orbeginning of the microchannel) better control clotting. Withoutintending to limit the invention to any particular mechanism, it isbelieved that the reagents in solution coming in from both sides createa type of barrier on the side walls of the microchannel, inhibitingcontact of the blood (or other input fluid) with the side walls. Thisinhibits clotting induced by contact with the walls of the microchannel.The blood (or other input fluid) travels down the microchannel to the“active” region, which may have cells (e.g. a monolayer of cells, suchas endothelial cells). In this manner, clotting caused by theinteraction of cells in the active region is distinguished fromnonspecific clotting induced by contact with the side walls of themicrochannel.

In some embodiments, it may be desirable to further treat the blood (orother fluid with blood components) as it leaves the active region of themicrochannel, or immediately thereafter, in order to reduce the chanceof clotting after testing. In one embodiment, the present inventioncontemplates one or more additive channels (positioned near an outputport) containing one or more reagents that will inactivate the nativecoagulation cascade in the blood that makes contact with it “on-chip” asit leaves the active or experimental region of the chip, permitting theblood to flow out the output port. While one additive channel can beused, it has been found empirically that two channels (one on eitherside of the output port or end of the microchannel) better controlclotting.

Therefore, the present invention contemplates a method of adding reagentto a fluid sample in a microfluidic device, comprising: a) providing i)a fluid sample comprising anticoagulant, and ii) a microfluidic devicecomprising one or more additive channels in fluidic communication withat least one microchannel, said one or more additive channels comprisingiii) a reagent solution comprising one or more reagents capable ofre-activating the coagulation cascade, and; b) introducing said fluidsample into said microchannel of said microfluidic device underconditions such that a portion of said fluid sample contacts saidreagent solution as said fluid sample moves through said microchannel.It is not intended that the present invention be limited to the type ornature of the fluid. In one embodiment, the fluid contains a componentor cell associated with clotting. In one embodiment, said fluid samplecomprises platelets. In one embodiment said fluid sample is blood (or ablood substitute). In one embodiment, said blood is human blood. It isnot intended that the present invention be limited by the type or natureof the anticoagulant. In one embodiment, said anticoagulant is sodiumcitrate. In one embodiment, said anticoagulant is ethylenediaminetetraacetic acid (EDTA). In one embodiment, said anticoagulant was addedto said human blood at the time it was collected from said human.

There are a variety of ways to introduce the fluid into themicrochannel. In one embodiment, said microfluidic device comprises aninput port in fluidic communication with said microchannel and saidintroducing of step b) is through said input port.

It is not intended that the present invention be limited by the numberor positioning of the additive channels. In one embodiment, said one ormore additive channels are positioned at or near said input port. In oneembodiment, a first additive channel is positioned on one side of saidmicrochannel near said input port. In one embodiment, a second additivechannel is positioned on another side of said microchannel near saidinput port.

It is not intended that the present invention be limited to how it isused. However, in a preferred embodiment, said at least one microchannelcomprises an active region comprising cells. In one embodiment, saidcells are living cells. In one embodiment, said cells are fixed cells.In one embodiment, said cells comprise endothelial cells. In oneembodiment, said endothelial cells are vascular endothelial cells. Inone embodiment, said vascular endothelial cells are a monolayer. In oneembodiment, said monolayer is disposed on a membrane. In one embodiment,said monolayer is attached to a cell adhesion promoting substance thatcoats the microchannel. In one embodiment, said cell adhesion promotingsubstance comprises one or more ECM proteins.

The present invention also contemplates, in one embodiment, a method ofadding reagent to a fluid sample in a microfluidic device, comprising:a) providing i) a fluid sample comprising a first anticoagulant, and ii)a microfluidic device comprising one or more first additive channels influidic communication with a first end of a microchannel, said one ormore first additive channels comprising iii) a first reagent solutioncomprising one or more reagents capable of re-activating the coagulationcascade, said microfluidic device further comprising one or more secondadditive channels in fluidic communication with a second end of amicrochannel, said one or more second additive channels comprising iv) asecond reagent solution comprising a second anticoagulant; b)introducing said fluid sample into said microchannel of saidmicrofluidic device under conditions such that a portion of said fluidsample contacts said first reagent solution as said fluid sample movesthrough said microchannel so as to create a treated portion; and c)contacting said treated portion with said second reagent solution. It isnot intended that the present invention be limited to the type or natureof the fluid. In one embodiment, the fluid contains a component or cellassociated with clotting. In one embodiment, said fluid sample comprisesplatelets. In one embodiment said fluid sample is blood (or a bloodsubstitute). In one embodiment, said blood is human blood. It is notintended that the present invention be limited by the type or nature ofthe anticoagulant. In one embodiment, said anticoagulant is sodiumcitrate. In one embodiment, said anticoagulant is ethylenediaminetetraacetic acid (EDTA). In one embodiment, said anticoagulant was addedto said human blood at the time it was collected from said human.

There are a variety of ways to introduce the fluid into the device. Inone embodiment, said microfluidic device comprises an input port influidic communication with said microchannel at said first end and saidintroducing of step b) is through said input port.

It is not intended that the present invention be limited by the numberof positioning of the additive channels. In one embodiment, said one ormore first additive channels are positioned at or near said input port.In one embodiment, one first additive channel is positioned on one sideof said microchannel near said input port. In one embodiment, anotherfirst additive channel is positioned on another side of saidmicrochannel near said input port.

A variety of different agents can be used. In one embodiment, said firstreagent solution comprises calcium and magnesium. In one embodiment,said second reagent solution comprises ethylenediamine tetraacetic acid(EDTA). In an alternative embodiment, an aqueous solution can be used toprevent coagulation (e.g. diluting blood with a saline solution or abuffered solution to prevent coagulation).

It is not intended that the present invention be limited as to the useof the device. However, in a preferred embodiment, said at least onemicrochannel comprises an active region comprising cells. A variety ofcell types are contemplated. In one embodiment, said cells are livingcells. In one embodiment, said cells are fixed cells. In one embodiment,said cells comprise endothelial cells. In one embodiment, saidendothelial cells are vascular endothelial cells. In one embodiment,said vascular endothelial cells are a monolayer. In one embodiment, saidmonolayer is disposed on a membrane. In one embodiment, said monolayeris attached to a cell adhesion promoting substance that coats themicrochannel. In one embodiment, said cell adhesion promoting substancecomprises one or more ECM proteins.

In a further embodiment, said microfluidic device comprises an outputport in fluidic communication with said microchannel at said second end.In one embodiment, said one or more second additive channels arepositioned at or near said output port. In one embodiment, one secondadditive channel is positioned on one side of said microchannel nearsaid output port.

In one embodiment, another first additive channel is positioned onanother side of said microchannel near said output port.

As noted above, the present invention contemplates methods, devices andsystems. In one embodiment, the present invention contemplates amicrofluidic device comprising i) a microchannel in fluidiccommunication with ii) an input port and iii) an output port, iv) one ormore first additive channels in fluidic communication with at least onemicrochannel, positioned at or near said input port. It is not intendedthat the device be limited to the positioning or number of additivechannels. In one embodiment, one first additive channel is positioned onone side of said microchannel near said input port. In one embodiment,another first additive channel is positioned on another side of saidmicrochannel near said input port. In one embodiment, the device furthercomprises v) one or more second additive channels in fluidiccommunication with said microchannel, positioned at or near said outputport. In one embodiment, one second additive channel is positioned onone side of said microchannel near said output port. In one embodiment,another second additive channel is positioned on another side of saidmicrochannel near said output port.

It is not intended that the use of the device be restricted. However, ina preferred embodiment, said microchannel comprises an active regioncomprising cells (whether viable or fixed), including but not limited tohuman cells (e.g. liver cells, lung cells, etc.).

In one embodiment, the present invention contemplates a system,comprising: a) a fluid sample comprising anticoagulant, said fluidsample disposed in b) a microfluidic device comprising i) a microchannelin fluidic communication with ii) an input port and iii) an output port,iv) one or more first additive channels in fluidic communication with atleast one microchannel, positioned at or near said input port, saidfirst additive channels comprising a first reagent solution comprisingone or more reagents capable of re-activating the coagulation cascade.In one embodiment, said microfluidic device further comprises v) one ormore second additive channels in fluidic communication with saidmicrochannel, positioned at or near said output port. In one embodiment,said fluid (whether blood, or merely containing some blood components)moves through the microchannel and come in contact with one or moreadditives via the fluidic communication of the microchannel with one ormore additive channels.

In one embodiment, the present invention contemplates devices with inputadditive channels, output additive channels or both. In one embodiment,the present invention contemplates a microfluidic device, comprising: aninput channel; an output channel; a test channel, wherein said testchannel comprises an input portion in fluidic communication with saidinput channel and an output portion in fluidic communication with saidoutput channel; (optionally) endothelial cells disposed within at leastone portion of said test channel; and an input additive channel, whereinsaid input additive channel is in fluidic communication with said inputportion of said test channel. In one embodiment said input channel andsaid input additive channel each have a fluidic resistance. It is notintended that, when cells are used in the device, that they be living.In one embodiment, said endothelial cells are living (e.g. viable) asmeasured by any technique (e.g. dye exclusion, biomarkers, secretedproteins, replication, etc.). In one embodiment, said endothelial cellsare fixed. In one embodiment, said input additive channel is configuredto deliver fluid to at least two opposing sides of said test channel(e.g. in a manner similar to that shown in FIG. 4A). In one embodiment,said input additive channel divides into two or more additive channelbranches, wherein said two or more additive channel branches areconfigured to produce an approximately equal fluidic resistance (e.g. sothat there is an approximately equal flow rate in said two or moreadditive channel branches). In one embodiment, the device furthercomprises an output additive channel in fluidic communication with saidoutput portion of said test channel. It is not intended that the presentinvention be limited to the design of the microfluidic device. In oneembodiment, the device further comprises a porous membrane and a backchannel, wherein said membrane is situated between at least one portionof said test channel and at least one portion of said back channel (e.g.in a manner similar to that shown in FIG. 2). In one embodiment, atleast one non-endothelial cell type is disposed within at least oneportion of said hack channel. In one embodiment, input channel furthercomprises a fluidic resistor (e.g. serpentine channels). In oneembodiment, said input additive channel further comprises a fluidicresistor. In one embodiment, said output additive channel furthercomprises a fluidic resistor. In one embodiment, the device furthercomprises at least one reservoir. The reservoir can be for the input,the output, or any of the additive channels. In a preferred embodiment,reservoirs for input additive channel or output additive channelsreagents are integrated on the microfluidic device (“on-chip”). In oneembodiment, the device further comprises an input reservoir in fluidiccommunication with said input channel. In one embodiment, the devicefurther comprises an input additive reservoir in fluidic communicationwith said input additive channel. In one embodiment, the device furthercomprises a pressure regulator, said pressure regulator adapted to applya pressure to both the input reservoir and the input additive reservoir.

The present invention also contemplates in another embodiment, amicrofluidic device, comprising: an input channel; an output channel; atest channel, wherein said test channel comprises an input portion influidic communication with said input channel and an output portion influidic communication with said output channel; (optionally) endothelialcells disposed within at least one portion of said test channel; and anoutput additive channel, wherein said output additive channel is influidic communication with said output portion of said test channel.Again, when used, the endothelial cells may be living or fixed. In oneembodiment, said output additive channel is configured to deliver fluidto at least two opposing sides of said test channel (e.g. in a mannersimilar to that shown in FIG. 12). In one embodiment, said outputadditive channel divides into two or more additive channel branches,wherein said two or more additive channel branches are configured toproduce an approximately equal fluidic resistance. In one embodiment,the device further comprises an input additive channel in fluidiccommunication with said input portion of said test channel. Again, it isnot intended that the present invention be limited by the design of themicrofluidic device. In one embodiment, the device further comprises aporous membrane and a back channel, wherein said membrane is situatedbetween at least one portion of said test channel and at least oneportion of said back channel. In one embodiment, at least onenon-endothelial cell type is disposed within at least one portion ofsaid back channel. In one embodiment, said input channel furthercomprises a fluidic resistor. In one embodiment, said output additivechannel further comprises a fluidic resistor. In one embodiment, saidinput additive channel further comprises a fluidic resistor. In oneembodiment, the device further comprises at least one reservoir. Thereservoir can be for the input, the output, or any of the additivechannels. In a preferred embodiment, reservoirs for input additivechannel or output additive channels reagents are integrated on themicrofluidic device (“on-chip”). In one embodiment, the device furthercomprises an input reservoir in fluidic communication with said inputchannel. In one embodiment, the device further comprises an outputadditive reservoir in fluidic communication with said output additivechannel. In one embodiment, the device further comprises a pressureregulator, said pressure regulator adapted to apply a pressure to boththe input reservoir and the output additive reservoir.

The present invention also contemplates methods of using additivechannels. In one embodiment, the present invention contemplates a methodof using a microfluidic device, comprising: a) providing, i) amicrofluidic device, comprising: an input channel, an output channel, atest channel, wherein said test channel comprises an input portion influidic communication with said input channel and an output portion influidic communication with said output channel, (optionally) endothelialcells disposed within at least one portion of said test channel; and aninput additive channel, wherein said input additive channel is influidic communication with said input portion of said test channel; ii)an anti-coagulated biological sample comprising cells, and iii) an agentthat restores the coagulation abilities of said biological sample; b)flowing said anti-coagulated biological sample into said input channeland into said input portion of said test channel; and c) flowing saidagent into said input additive channel under conditions such that agentcontacts at least a portion of said anti-coagulated biological sample,wherein steps b) and c) can be performed in any order or simultaneously.In one embodiment, step b) is done before step c). In one embodimentstep b) is done after step c). In one embodiment, steps b) and c) areperformed simultaneously. The flow rates in steps b) and c) can be, butneed not be, the same. In one embodiment, said flowing of step b) isdone at first flow rate, and wherein said flowing of step c) is done ata second flow rate, wherein the first and second flow rates areproportional to each other. In one embodiment, the flow rate of step c)is a fraction (e.g. one quarter, one half, etc.) of the flow rate ofstep b). In one embodiment, the flow rates are chosen so that the amountof agent mixed in is sufficient to restore the coagulation abilities ofsaid biological sample. In one embodiment, said contacting in step c)allows for a thrombotic process (e.g. such that another component orcondition might initiate a thrombotic process). In one embodiment, themethod further comprises d) optically observing said thrombotic process.It is not intended that the present invention be limited to the natureof the biological sample. In one embodiment, said biological samplecomprises blood. In one embodiment, said biological sample comprises atleast one blood component (e.g. platelets, red blood cells, white bloodcells, etc.). In one embodiment, said agent that restores thecoagulation abilities comprises calcium. In one embodiment, saidoptically observing comprises live-cell imaging. In one embodiment, saidoptically observing comprises live-cell imaging during said flowing ofsaid biological sample. In one embodiment, the method further comprisesa step of fixing said cells after step c). In one embodiment, the methodfurther comprises a step of fixing said cells before step d). In oneembodiment, at least a portion of said anti-coagulated biological sampleflows out said output channel. In one embodiment, the method furthercomprises the step of collecting at least a portion of said sample fromthe output channel. In one embodiment, the method further comprises thestep of analyzing said sample collected from said output channel. In oneembodiment, said analyzing comprises testing for the existence of, orthe amount of, components in said sample collected from said outputchannel. In one embodiment, said components are selected from the groupconsisting of cytokines, antibodies, blood cells, cell surface markers,proteins, RNA (including micro-RNA), DNA, biomarkers and clottingfactors. In one embodiment, said device further comprises at least oneoutput additive channel in fluidic communication with said outputportion of said test channel. In one embodiment, the present inventioncontemplates the testing of drugs, candidate drugs or other compounds.In one embodiment, the method further comprises adding a test a compoundto the agent before step c). In one embodiment, the method furthercomprises adding a test a compound to the biological sample before orduring step b). In one embodiment, the test compound is evaluated forthe potential to initiate, cause or otherwise enable a thromboticprocess. For example, the test compound might be evaluated for thepotential to promote on adhesion of platelets and/or proteins to asurface. On the other band, the test compound might be evaluated for thepotential to promote platelet activation and/or aggregation. In oneembodiment, the test compound is evaluated for the potential to inhibit,block or otherwise interfere with a thrombotic process. For example, atest compound might be evaluated for the potential to inhibit adhesionof platelets and/or proteins to a surface. On the other hand, the testcompound might be evaluated for the potential to inhibit plateletactivation and/or aggregation. Still further, the test compound isevaluated for safety or efficacy. In one embodiment, the presentinvention contemplates comparing measures of thrombosis at differentconcentrations of the said test compound (including testing with andwithout the compound). In one embodiment, first and second testcompounds are evaluated (e.g. for their ability to work together, workagainst one another, work synergistically, etc.). In one embodiment of atwo compound method, a first compound is employed to induce coagulationand the second compound is employed in an attempt to stop it or at leastinhibit it. In another embodiment of a two compound method, a firstcompound creates a disease model and a second compound is the one underinvestigation to treat the disease. Again, it is not intended that thepresent invention be limited only to specific microfluidic designs. Inone embodiment, said microfluidic device further comprises a porousmembrane and a back channel, wherein said membrane is situated betweenat least one portion of said test channel and at least one portion ofsaid back channel. In one embodiment, at least one non-endothelial celltype is disposed within at least one portion of said back channel. Inone embodiment, the method further comprises analyzing at least some ofsaid cells of at least one non-endothelial cell type after step c). Inone embodiment, the method further comprises d) flowing a third fluidinto said back channel. In one embodiment, the method further comprisesanalyzing the outflow of said back channel.

In still another embodiment, the present invention contemplates a methodof using a microfluidic device, comprising: a) providing i) amicrofluidic device, comprising: an input channel, an output channel, atest channel, wherein said test channel comprises an input portion influidic communication with said input channel and an output portion influidic communication with said output channel, (optionally) endothelialcells disposed within at least one portion of said test channel; and anoutput additive channel, wherein said output additive channel is influidic communication with said output portion of said test channel; ii)a biological sample, and iii) an anti-coagulation agent, and b) flowingsaid biological sample into said input channel, into said input portionof said test channel, and into said output portion of said test channel;and c) flowing said agent into said output additive channel underconditions such that agent contacts at least a portion of saidbiological sample, wherein steps b) and c) can be performed in any orderor simultaneously. In one embodiment, step b) is done before step c). Inone embodiment step b) is done after step c). In one embodiment, stepsb) and c) are performed simultaneously. The flow rates in steps b) andc) can be, but need not be, the same. In one embodiment, said flowing ofstep b) is done at first flow rate, and wherein said flowing of step c)is done at a second flow rate, wherein the first and second flow ratesare proportional to each other. In one embodiment, the flow rate of stepc) is a fraction (e.g. one quarter, one half, etc.) of the flow rate ofstep b). In one embodiment, the flow rates are chosen so that the amountof agent mixed in is sufficient to restore the coagulation abilities ofsaid biological sample. In one embodiment, said output channel and saidoutput additive channel each have a fluidic resistance. In oneembodiment, the fluidic resistance of said output additive channel isadapted with respect to the fluidic resistance of said input channel(e.g. to be proportional to the input channel). In one embodiment, saidcontacting in step c) allows for a thrombotic process (e.g. such thatanother component or condition might initiate a thrombotic process). Inone embodiment, the method further comprises d) optically observing saidthrombotic process. In one embodiment, said biological sample comprisesblood. In one embodiment, said biological sample comprises at least oneblood component (e.g. platelets, red blood cells, white blood cells,etc.). In one embodiment, said agent is selected from the groupconsisting of EDTA, citrate, ACD, heparin and coumarin. In oneembodiment, said optically observing comprises live-cell imaging. In oneembodiment, said optically observing comprises live-cell imaging duringsaid flowing of said biological sample. In one embodiment, the methodfurther comprising a step of fixing said cells after step c). In oneembodiment, the method further comprises a step of fixing said cellsbefore step d). In one embodiment, at least a portion of said biologicalsample flows out said output channel. In one embodiment, the methodfurther comprises the step of collecting at least a portion of saidsample from the output channel. In one embodiment, the method furthercomprises the step of analyzing said sample collected from said outputchannel. In one embodiment, said analyzing comprises testing for theexistence of, or the amount of, components in said sample collected fromsaid output channel. In one embodiment, said components are selectedfrom the group consisting of cytokines, antibodies, blood cells, cellsurface markers, proteins, RNA (including micro-RNA), DNA, biomarkersand clotting factors. In one embodiment, said device further comprisesat least one input additive channel. In one embodiment, the presentinvention contemplates the testing of drugs, candidate drugs or othercompounds. In one embodiment, the method further comprises adding a testa compound to the agent before step c). In one embodiment, the methodfurther comprises adding a test a compound to the biological samplebefore or during step b). In one embodiment, the test compound isevaluated for the potential to initiate, cause or otherwise enable athrombotic process. For example, the test compound might be evaluatedfor the potential to promote the adhesion of platelets and/or proteinsto a surface. On the other hand, the test compound might be evaluatedfor the potential to promote platelet activation and/or aggregation. Inone embodiment, the test compound is evaluated for the potential toinhibit, block or otherwise interfere with a thrombotic process. Forexample, a test compound might be evaluated for the potential to inhibitadhesion of platelets and/or proteins to a surface. On the other hand,the test compound might be evaluated for the potential to inhibitplatelet activation and/or aggregation. Still further, the test compoundis evaluated for safety or efficacy. In one embodiment, the presentinvention contemplates comparing measures of thrombosis at differentconcentrations of the said test compound (including testing with andwithout the compound). In one embodiment, first and second testcompounds are evaluated (e.g. for their ability to work together, workagainst one another, work synergistically, etc.). In one embodiment of atwo compound method, a first compound is employed to induce coagulationand the second compound is employed in an attempt to stop it or at leastinhibit it. In another embodiment of a two compound method, a firstcompound creates a disease model and a second compound is the one underinvestigation to treat the disease. Again, it is not intended that thepresent invention be limited only to specific microfluidic designs. Inone embodiment, said microfluidic device further comprises a porousmembrane and a back channel, wherein said membrane is situated betweenat least one portion of said test channel and at least one portion ofsaid back channel. In one embodiment, at least one non-endothelial celltype is disposed within at least one portion of said back channel. Inone embodiment, the method further comprises analyzing at least some ofsaid cells of at least one non-endothelial cell type after step c). Inone embodiment, the method further comprises d) flowing a third fluidinto said back channel so as to create an outflow of said back channel.In one embodiment, the method further comprises analyzing the outflow ofsaid back channel.

The present invention also contemplates systems comprising additivechannels. In one embodiment, the present invention contemplates a systemcomprising: a) a microfluidic device comprising: an input channel; anoutput channel; a test channel, wherein said test channel comprises aninput portion in fluidic communication with said input channel and anoutput portion in fluidic communication with said output channel;(optionally) endothelial cells disposed within at least one portion ofsaid test channel; and an input additive channel, wherein said inputadditive channel is in fluidic communication with said input portion ofsaid test channel; b) an input channel reservoir in fluidiccommunication with said input channel; c) an input additive channelreservoir in fluidic communication with said input additive channel; andd) a pressure source configured to apply pressure to both said inputchannel reservoir and said input additive channel reservoir. In oneembodiment, said input additive channel is configured to provide afluidic resistance that is proportional to the fluidic resistance ofsaid input channel. In one embodiment, said input additive channelcomprises a first fluidic resistor, and said input channel comprises asecond fluidic resistor. In one embodiment, it is contemplated that thesingle pressure source can create the correct ratio of flow ratesbetween the input channel and the additive channel. In a preferredembodiment, reservoirs are integrated on the microfluidic device(“on-chip”).

In yet another embodiment, the present invention contemplates a systemcomprising: a) a microfluidic device comprising: an input channel; anoutput channel; a test channel, wherein said test channel comprises aninput portion in fluidic communication with said input channel and anoutput portion in fluidic communication with said output channel;(optionally) endothelial cells disposed within at least one portion ofsaid test channel; and an output additive channel, wherein said outputadditive channel is in fluidic communication with said output portion ofsaid test channel; b) an input channel reservoir in fluidiccommunication with said input channel; c) output additive channelreservoir in fluidic communication with said output additive channel;and d) a pressure source adapted to apply pressure to both said inputchannel reservoir and said output additive channel reservoir. In oneembodiment, said output additive channel is configured to provide afluidic resistance that is proportional to the fluidic resistance ofsaid input channel. In one embodiment, said output additive channelcomprises a first fluidic resistor, and input channel comprises a secondfluidic resistor. In a preferred embodiment, reservoirs are integratedon the microfluidic device (“on-chip”).

In yet another embodiment, the present invention contemplates a system,comprising i) a plurality of microfluidic devices (or simplymicrofluidic channels) sharing a single additive port between saidplurality of said devices (or microfluidic channels), wherein saidsingle additive port has a plurality of tubular branches, wherein eachsaid branch is a fluidic connection with one device (or one channel),and wherein each said branch has an additive fluidic flow rate, and(optionally) ii) a plurality of fluidic flow resistors, wherein eachbranch has at least one resistor configured for controlling an additivefluidic flow rate.

In still another embodiment, the present invention contemplates a systemcomprising two (or more) constructs, each construct comprising: an inputchannel, an output channel, a test channel, wherein said test channelcomprises an input portion in fluidic communication with said inputchannel and an output portion in fluidic communication with said outputchannel, (optionally) endothelial cells disposed within at least oneportion of said test channel; an input additive channel, wherein saidinput additive channel is in fluidic communication with said inputportion of said test channel, wherein the input additive channel of saidfirst construct and the input additive channel of said second constructare fluidically coupled to a common additive channel. In one embodiment,the fluidic resistance of said input additive channel of first constructand said input additive channel of said second construct are adapted forapproximately equal fluidic resistance. In one embodiment, the inputadditive channel of said first construct further comprises a firstfluidic resistor, and wherein the input additive channel of said secondconstruct further comprises a second fluidic resistor. In oneembodiment, said constructs are microfluidic devices. In one embodiment,the system further comprises a cell-seeding channel, said cell-seedingchannel fluidically coupled to said test channel of first construct andsaid test channel of second construct. In a method for using thissystem, the present invention contemplates an embodiment wherein thecell-seeding channel is used to seed both constructs (e.g. at once priorto an experiment).

In still another embodiment, the present invention contemplates a systemcomprising two (or more) constructs, each construct comprising: an inputchannel, an output channel, a test channel, wherein said test channelcomprises an input portion in fluidic communication with said inputchannel and an output portion in fluidic communication with said outputchannel, (optionally) endothelial cells disposed within at least oneportion of said test channel; an output additive channel, wherein saidoutput additive channel is in fluidic communication with said outputportion of said test channel, wherein the output additive channel ofsaid first construct and the output additive channel of said secondconstruct are fluidically coupled to a common additive channel. In oneembodiment, the fluidic resistance of said output additive channel offirst construct and said output additive channel of said secondconstruct are adapted for approximately equal fluidic resistance. In oneembodiment, the output additive channel of said first construct furthercomprises a first fluidic resistor, and wherein the output additivechannel of said second construct further comprises a second fluidicresistor. In one embodiment, said constructs are microfluidic devices.In one embodiment, the system further comprises a cell-seeding channel,said cell-seeding channel fluidically coupled to said test channel offirst construct and said test channel of second construct. In a methodfor using this system, the present invention contemplates an embodimentwherein the cell-seeding channel is used to seed both constructs (e.g.at once prior to an experiment).

The present invention contemplates in any of the above-described systemsthat the microfluidic devices (or plurality of microfluidic devices) orchannels (or plurality of channels) comprise active regions withinviewing range of a microscope. Alternatively, said microfluidic devices(or plurality of microfluidic devices) or channels (or plurality ofchannels) comprise active regions within a single field of view of amicroscope image.

The present invention also contemplates a method comprising: a)providing: i) a microfluidic device comprising two or more testchannels, wherein each said test channel comprises cells (e.g.endothelial cells); ii) at least one biological sample; and iii) amicroscope; b) flowing said at least one biological sample into said twoor more said test channels under conditions that initiate thrombusformation in at least one of said test channels; and c) imaging saidtest channels using said microscope. In one embodiment, step b)comprises flowing one of said at least one biological samples into twoor more said test channels. For example, the same blood can be flowedinto several channels, or alternatively, each channel can get its ownblood sample. In one embodiment, said at least one test channel of saidmicrofluidic device further comprises: an input channel and an inputportion of said test channel, wherein said input portion is in fluidiccommunication with said input channel, and an input additive channel,wherein said input additive channel is in fluidic communication withsaid input portion of said test channel. In one embodiment, said atleast one test channel of said microfluidic device further comprises: anoutput channel and an output portion of said test channel, wherein saidinput portion is in fluidic communication with said output channel, andan output additive channel, wherein said input additive channel is influidic communication with said output portion of said test channel. Inone embodiment, said imaging of step c) comprises imaging at least aportion of each test channel of said microfluidic device in a singlemicroscope field. In one embodiment, said microscope further comprises amicroscope stage, and wherein imaging of step c) comprises imaging atleast a portion of each test channel of said microfluidic device bymeans of motion of said microscope stage.

The present invention also contemplates in one embodiment a microfluidicdevice comprising a test channel, and endothelial cells disposed withinat least a portion of said test channel, wherein said test channelincludes at least one geometrical feature selected from the listconsisting of a gradual change of cross-section, an abrupt change ofcross-section, a bend, a bifurcation. In one embodiment, themicrofluidic device further comprises an input channel, an input portionof said test channel, wherein said input portion is in fluidiccommunication with said input channel, and an input additive channel,wherein said input additive channel is in fluidic communication withsaid input portion of said test channel. In one embodiment, said atleast one test channel of said microfluidic device further comprises: anoutput channel, an output portion of said test channel, wherein saidinput portion is in fluidic communication with said output channel, andan output additive channel, wherein said input additive channel is influidic. communication with said output portion of said test channel.

Definitions

Anticoagulants are used to prevent clot formation both in vitro and invivo. In the specific field of in vitro diagnostics, anticoagulants arecommonly added to collection tubes either to maintain blood in the fluidstate for hematological testing or to obtain suitable plasma forcoagulation and clinical chemistry analyses.

Calcium is necessary for a wide range of enzyme reactions of thecoagulation cascade and its removal prevents blood clotting within thecollection tube. Ethylenediamine tetraacetic acid (EDTA) is a polyproticacid containing four carboxylic acid groups and two amine groups withlone-pair electrons that chelate calcium and several other metal ions.Historically, EDTA has been recommended as the anticoagulant of choicefor hematological testing because it allows the best preservation ofcellular components and morphology of blood cells. The remarkableexpansion in laboratory test volume and complexity over recent decadeshas amplified the potential spectrum of applications for thisanticoagulant, which can be used to stabilize blood for a variety oftraditional and innovative tests.

One can also anti-coagulate blood with sodium citrate (e.g. 3.2%). EDTAand sodium citrate are both calcium chelators. Without wishing to bebound by theory, platelet function may depend upon the presence of Ca²⁺and Mg²⁺. Thus, for a fluid sample comprising a citrated blood sample(where titration of a blood sample generally quenches the free Ca²⁺ andMg²⁺ ions to prevent blood coagulation), addition of Ca²⁺ (e.g., calciumchloride) and Mg²⁺ (magnesium chloride) to the fluid sample can helprestore the native physiological state of the platelet, e.g., to allowplatelet aggregation or coagulation. Thus, in some embodiments, thecitrated blood sample can be added with Ca²⁺ (e.g., calcium chloride)and Mg²⁺ (magnesium chloride) such that the final concentrations reachabout 4-12 mM and 3-10 mM, respectively.

In an alternative embodiment, an aqueous solution can be used to preventcoagulation (e.g. diluting blood with a saline solution or a bufferedsolution to prevent coagulation).

In one embodiment, blood (or other fluid sample with blood components)is introduced into the microfluidic device comprising on or morechannels, and more specifically, one or more microchannels. The surfaceover which the sample flows to perform the cell analysis using themethods described herein can be a surface of any material that iscompatible to the fluid sample and cells. Exemplary materials for thefluid-contact surface can comprise glass, synthetic polymers (e.g.,PDMS, polysulfonate, and polycarbonate), hydrogels, and a combinationthereof.

“Channels” are pathways (whether straight, curved, single, multiple, ina network, etc.) through a medium (e.g., silicon, glass, polymer, etc.)that allow for movement of liquids and gasses. In some embodiments,described herein “test channel” are used and these need not have thesame shape throughout their length. For example, one can change thechannel cross-section (expansions and contractions), one can bend thechannel (including a spiral version), and/or one can bifurcate thechannel (include the corner areas). Channels can connect or be coupledwith other components, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, liquid-intake ports and gas vents, Channels can also permit on-chipmixing of cells with reagents, such as reagents that re-activate thecoagulation cascade and anticoagulants. Microchannels are channels withdimensions less than 1 millimeter and greater than 1 micron. It is notintended that the present invention be limited to only certainmicrochannel geometries. In one embodiment, a four-sided microchannel iscontemplated. In another embodiment, the microchannel is circular (inthe manner of a tube) with curved walls. In yet another embodiment,combination of circular or straight walls are used.

One portion of a microchannel can be a membrane. For example, the floorof a microchannel can comprise a membrane, including a porous membrane.The microchannel (or portion thereof) or membrane can be coated withsubstances such as various cell adhesion promoting substances or ECMproteins, such as fibronectin, laminin or various collagen types orcombinations thereof. For example, endothelial cells can attach to acollagen coated microchannel.

It is not intended that the present invention be limited by the numberor nature of channels in the microfluidic device. In some embodiments,the surface can be a surface of a fluid-flowing conduit or passagewaydisposed in a solid substrate. In some embodiments, the surface can be asolid surface. For example, in one embodiment, the solid surface can bea wall surface of a fluid channel, e.g., a microfluidic channel.

Additionally, the term “microfluidic” as used herein relates tocomponents where moving fluid is constrained in or directed through oneor more channels wherein one or more dimensions are 1 mm or smaller(microscale). Microfluidic channels may be larger than microscale in oneor more directions, though the channels) will be on the microscale in atleast one direction. In some instances the geometry of a microfluidicchannel may be configured to control the fluid flow rate through thechannel (e.g. increase channel height to reduce shear). Microfluidicchannels can be formed of various geometries to facilitate a wide rangeof flow rates through the channels.

In some embodiments, fluids comprising platelets are introduced into themicrofluidic device in order to detect platelet function or dysfunction.As used herein, the term “platelet dysfunction” refers to abnormalplatelet behavior, as compared to healthy platelets. In one embodiment,platelet dysfunction can be caused by increased adhesion to anendothelium (e.g., by at least about 30% or more), as compared tohealthy platelets. In one embodiment, platelet dysfunction can be causedby abnormal detachment from other platelets and/or from an endothelium(e.g., by at least about 30% or more), as compared to healthy platelets.In one embodiment, platelet dysfunction can be caused by abnormaltranslocation (e.g., by at least about 30% or more), as compared tohealthy platelets. As used herein, the term “abnormal translocation”refers to a platelet that gets activated in one location and deposits atanother location to form a clot or cause inflammation response. Forexample, thromboembolism can be considered as abnormal translocation. Inone embodiment, platelet dysfunction can be caused by increasedaggregation between platelets (e.g., by at least about 30% or more), ascompared to healthy platelets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a perspective view of one embodiment of amicrofluidic device or chip showing input and output ports in fluidiccommunication with an active region or experimental region.

FIG. 2 illustrates an exploded view of the microfluidic device of FIG. 1

FIGS. 3A-C shows one embodiment of on-chip mixing using a single input,i.e. a single additive channel in fluidic communication. FIG. 3A showsthe input channel attached to the microfluidic port of the microfluidicdevice. FIGS. 3B and 3C are photographs showing the single input streamand single input mixing, respectively.

FIGS. 4A-C shows one embodiment of on-chip mixing using a dual input,i.e. two additive channels in fluidic communication. FIG. 4A shows aschematic of the dual input additive channels attached to themicrofluidic port of the microfluidic device. FIG. 4B and FIG. 4C arephotographs showing the dual input streams and dual input mixing,respectively.

FIGS. 5A-B shows exemplary illustrations of photographs demonstratingon-chip mixing of anticoagulant. FIG. 5A shows tubing connecting asource of sodium citrate to the microfluidic device (not shown). On-chipmixing with sodium citrate (arrow) allows samples to flow freely, whilelack of anticoagulant input clogs collection tubes and can slow orcompletely stop flow. FIG. 5B shows five tubes, four of which weretreated on-chip with anticoagulant and can be analyzed. Tube 5 was nottreated and contains a solid mass of coagulated blood, which cannot beused for testing.

FIGS. 6A-E shows exemplary illustrations of photographs of blood sampledfrom the outlet of standard chips (without anticoagulant) compared toanticoagulant added to disclosed chips through anticoagulant ports.Droplets of blood sampled from the effluents at the end of a 15-minuteexperiment were absorbed on sterile paper then deposited on glass-slidefor further fluorescent microscopy imaging. Two representative imagesare shown here for each treatment. Platelets were labeled withCD41-TRITC antibody. FIG. 6A is a photograph of blood sampled from theeffluents (i.e. outlet) of standard chips without anticoagulant (bottomimage) or from the effluents of the disclosed chips equipped with theanticoagulant port and with anticoagulant (top image). FIG. 6B and FIG.6C are photographic images showing fluorescently-labeled plateletaggregates, demonstrating clotting in the untreated sample.Magnification ×10. FIG. 60 and FIG. 6E are photographic images for thetreated sample, with labeled platelets from the treated sample that aredispersed in the blood liquid phase, demonstrating an uncoagulatedstate. Magnification ×10.

FIG. 7A is a clot size analysis of platelet aggregates that occurred inthe blood during testing, demonstrating the distribution of sizes thatoccur during testing. FIG. 7B is the determination ofthrombin/anti-thrombin (TAT) complex concentration in blood aftertreatment with pro-coagulant factors.

FIG. 8 is a schematic showing an additive channel with ridges forenhanced mixing. In this embodiment, a three-dimensional twisting flowis generated in the mixing channel with obliquely oriented ridges on onewall.

FIG. 9 is a schematic showing an additive channel with a staggeredherringbone design of ridges for enhanced mixing. In this embodiment, amixing cycle is composed of two sequential regions of ridges; thedirection of asymmetry of the herringbones switches with respect to thecenterline of the channel from one region to the next. The streamlinesof the flow it in the cross section are shown schematically above thechannel.

FIGS. 10A-D shows schematics depicting platelet thrombus formation overa monolayer of living endothelium. In a microchannel covered on allsides with untreated living endothelium (FIG. 10A), whole blood flowswithout clotting (FIG. 10B). In contrast, platelet-rich thrombus forms(FIG. 10C) if the endothelium is prestimulated by a pro-inflammatorycytokine, such as TNF-alpha, due to expression of procoagulatoryproteins at its surface (FIG. 10D). In some embodiments, the responsesof blood under flow shown in the figures can be reconstituted usingsimilar microchannels that are lined by a chemically preserved (e.g.fixed) endothelium.

FIG. 11 is a diagram of a simple microfluidic device or chip showinginlet and outlet ports in fluidic communication with a singlemicrochannel, with the active region or experimental region of thechannel highlighted (dashed line).

FIG. 12 is a schematic of one embodiment of a microfluidic deviceshowing the end of the microchannel and outlet port in fluidiccommunication with two additive channels comprising anticoagulant, sothat the sample leaving the microchannel remains fluid as it approachesthe outlet port and exits the microfluidic device.

FIG. 13 is a schematic of a partial top view of one embodiment of amicrofluidic device showing one end of the microchannel terminating at afirst port, the microchannel in fluidic communication with two additivechannels (one on either side) connecting to a second port (e.g. foradding the additive).

FIG. 14 is a schematic of a complete top view of one embodiment of amicrofluidic device showing two microchannels aligned with one another(i.e. the main body of the first channel is above the main body of thesecond channel), where each end of each microchannel terminates at aport, wherein each microchannel is in fluidic communication with twoadditive channels (one on either side just prior to the port), eachadditive channel connecting to separate port (e.g. for adding theadditive).

FIG. 15 is a schematic side view of the embodiment of a microfluidicdevice shown in FIG. 14, where each of two microchannels terminates at aport, each microchannel in fluidic communication with two additivechannels (one on either side near the port), each additive channelconnecting to a separate port (e.g. for adding the additive).

FIG. 16 is a schematic top view of one embodiment of a microfluidicdevice having four microchannels in parallel, where each end of eachmicrochannel terminates at a port, wherein each microchannel is influidic communication with two additive channels (one on either sidejust prior to the port), each additive channel connecting to separateport (e.g. for adding the additive).

FIG. 17 is a schematic side view of the embodiment of a microfluidicdevice shown in FIG. 16, where each of four microchannels have first andsecond ports, wherein each microchannel is in fluidic communication withtwo additive channels (one on either side just prior to the port), eachadditive channel connecting to separate port (e.g. for adding theadditive).

FIGS. 18A1-A2 through FIG. 18E-1 and FIG. 18E-2 demonstrates oneembodiment of an On-chip reconstitution of thrombosis showing schematicillustrations of an exemplary embodiment for a microfluidic-chip,micrographs of cells and charts comparing blood clotting events inducedby several compounds. FIG. 18A-1 is a schematic representation of oneembodiment of a Thrombosis-On-Chip (200), FIG. 18A-2 is a schematicrepresentation of the chip (200) showing the main features: inlet port(1810), main channel and imaged area (1820), outlet port (1815), and theon-chip anticoagulant port. FIG. 18B Top shows endothelial morphology byfluorescent VE-cadherin staining (stain as white lines). FIG. 18B Bottomshows a high magnification section of endothelial cells stained forVE-cadherin. FIG. 18C shows that under control conditions (whole bloodalone) platelets and fibrin shown as white spots and line, are sparseand detectable at the edge of the imaged areas. Endothelial exposure toTNF-α or pre-incubation of blood with soluble collagen (sCollagen) ledto formation of a high number of larger aggregates containing a plateletrich core decorated with fibrin. FIG. 18D shows scanning electronmicrographs of cells showing typical ultrastructure of blood clotsformed on-chip, in control conditions (‘Blood’) the sparse platelets aredispersed on an endothelial surface, in stimulated conditions (‘TNF-α’and ‘sCollagen’) activated platelet aggregates and fibrin networks withtrapped red blood cells are attached to an endothelial cell surface.FIG. 18E-1 shows platelet coverage and FIG. 18E-2 shows fibrindeposition (both charts using the lower treatment key shown in FIG.18E-2) that were significantly increased in stimulated plateletcoverage; fibrin deposition were significantly increased in stimulated,pro-thrombotic conditions in multiple donors (n=4, S.E.M., *p<0.05, nsnot significant), and the effects were suppressed by adding the drugEptifibatide. Overall, there is a highly significant difference betweenthe TNF-alpha treatment with and without Eptifibatide and betweensCollogen treatment with and without Eptifibatide.

FIGS. 19A-D show schematic illustrations of one embodiment ofmicrofluidic chips as a Thrombosis-On-Chip, micrographs of cells and achart showing vascular leakage values representing tissue integrity.FIG. 19A Left, schematic illustration of a cross-section of oneembodiment of a chip with an anticoagulant port used to monitorendothelial integrity. Right, endothelial cells covering the entiresurface of the vascular compartment were monitored via light microscopyimaging for 6 days. FIG. 19B shows tissue integrity was monitored via avascular leakage assay using fluorescent dextrin (3 Kda). FIG. 19C showsa schematic of one embodiment of a chip 1900 with an anticoagulant portattached to the top microfluidic channel where the upper channel is alsomarked with an OUTLET at one end. In other embodiments, such asdescribed in FIG. 19A, the lower vascular chamber has an anticoagulantport near the vascular outlet, shown in FIG. 14 and FIG. 15. FIG. 19Chas arrows pointing to INLET ports 1910 and 1911. The upper channel 1912emerges from one INLET 1910. The lower channel 1914 emerges fromunderneath the upper channel attached to the lower Inlet 1911. Arrowspoint to OUTLET ports 1915 and 1917. An arrow points to the IMAGING area(active region) 1920 outlined with dotted lines. An arrow points to theANTICOAGULANT port 1930. There are also arrows pointing to an additivechannel 1932 and 1934 surrounding the OUTLET port 1917. FIG. 19D showsrepresentative images obtained from the central area of the vascularchamber after blood perfusion (15 mins). The effects of the plateletinhibitor Eptifibatide are clearly visible in both conditions.

FIGS. 20A-B show exemplary micrographs of endothelium immunostained withanti-ICAM1 antibodies demonstrating exemplary expression of ICAM1 thatwas higher in INF-α stimulated endothelium than the control cells. FIG.20A shows immunostaining of healthy endothelium. ICAM1 is shown withexamples identified by white arrows. One example of a DAN stained nucleiis shown inside of a white circle. FIG. 20B shows immunostaining ofhealthy endothelium stimulated with TNF-α showing higher numbers ofcells identified by costaining with ICAM and DAPI (arrows).

FIGS. 21A-E show exemplary schematic illustrations, micrographs andcharts showing the combination of hu5c8 and sCD40L immune-complex(IC_(5c8)) induced thrombosis on-chip. FIG. 21A shows a schematicoverview of an embodiment showing how hu5c8 and sCD40L form immunecomplexes that in turn can activate platelets by engaging with the Fcγreceptors on their surface. FIG. 21B shows a colored micrograph ofimmunostained cells showing blood samples treated with combinedhu5C8/sCD40L promotes formation of sparse microthrombi (blood clots)rich in fibrin, platelets (large round circles) and fibrin (fibers).FIG. 21C shows that combined Hu5C8/sCD40L promotes formation of bloodclots constituted by small platelet aggregates and fibrin. FIG. 21Bshows platelet coverage measured on-chip after 12 minutes of continuousblood perfusion in presence of sCD40L, hu5c8 or combined hu5C8/sCD40Land normalized in respect to blood alone (n=4, bars indicate SEM, pvalue calculated using one-way ANOVA, ns: not significant). FIG. 21Eshows gene expression data obtained from chips treated with blood aloneor in combination with sCD40L and hu5c8 and normalized in respect tocells perfused with standard cell culture medium.

FIGS. 22A-B show one embodiment of a Vessel-On-Chip, where out-flowingblood allows sampling and analysis of effluent in addition to a chartdemonstrating comparative TAT levels following several differenttreatments. FIG. 22A, a citrate solution (from additive channels 2007and 2008) is actively pushed into the outflow stream (2010) of blood asit leaves the Vessel-On-Chip. This prevents clotting inside connectorsand tubing, allowing for longer experiments as well as conventionalanalysis of the out flowing blood samples. FIG. 22B, ThrombinAnti-Thrombin (TAT) levels are analyzed in plasma from blood flowing outof the vessel-on-chip device, TNF-α pre-treatment of the endothelium, aswell as sCollagen, or a combination of sCD40L and hu5c8 dosing in theblood before perfusion, leads to elevated levels of TAT in the outflowsamples as measured by ELIZA.

FIG. 23 shows an exemplary micrograph obtained from a moviedemonstrating 3D reconstruction of IC-induced Clot on Chip. A typicalblood clot induced by IC5c8 treatment includes nucleate cells andmicrothrombi (with platelets) trapped within the fibrin meshwork. Whenthis figure is in color: nucleate cells are DAPI stained colored purple;fibrin is colored cyan and platelets are colored yellow.

FIG. 24 shows a chart comparing gene expression data obtained from chipstreated with blood alone or in combination with sCD40L and hu5c8 andnormalized in respect to cells perfused with standard cell culturemedium. In particular, PAF1 (polymerase associated factor) and CD40 showhigher expression in blood treated with sCD40L and hu5c8 treated blood.Other genes measured included vWF (Von Willebrand Factor), SERPINE2(Serpin Family E Member 2), and PECAM1 (Platelet And Endothelial CellAdhesion Molecule 1).

FIGS. 25A-D reveal exemplary mechanistic insights into the thrombosisinduced by hu5C8/sCD40L-combined on-chip, specifically comparingtreatments of sCD40L; IC_(5C8); IC_(IV.3); and IC_(IgGσ). FIG. 25ASchematic representations of embodiments showing how antibodies withdifferent structures contribute and interfere with the interaction ofplatelets with immune complexes. By using antibodies with modified Fcregions (IgGσ, middle), or by using anti-Fcγ Receptor blockingantibodies (IV.3, right), immune complex interaction with plateletsshould be prevented. Key: large round circles represent quiescentplatelets, hearts represent sCD40L, Y represents Hu5c8, rounded narrowrectangles represent FCgRIIA, stars represent activated platelets, and aheart surrounded by Y's represents immune-complexes. Immune complexes(IC) formed with Hu5C8, but not with other treatments, and incubated inblood in the absence of blocking antibodies induced platelet adhesionFIG. 25B; FIG. 25C fibrin formation; and FIG. 25D TAT release on-chip.(n=15; n=5; n=7, respectively).

FIGS. 26A-C show exemplary charts comparing untreated blood totreatments of sCD40L; IC_(5C8); IC_(IV.3); and IC_(IgGσ), as in theprevious figure. FIG. 26A shows platelet coverage, as fold incrementincreases in respect to untreated blood; FIG. 26B shows fibrinfluorescence, as fold increment increases in respect to untreated blood;and FIG. 26C shows changes in TAT levels (ng/ml (ELIZA).

FIGS. 27A-E show exemplary charts, a stained image of blood clots andscanning electron micrographs comparing treatments of blood to untreatedblood. FIG. 27A shows an exemplary chart showing platelet coverage overtime (minutes) up to at least 12 minutes, comparing control blood tosCD40L, Hu5c8 and IC; FIG. 27B shows an exemplary chart demonstratingfold increment increases, in respect to untreated blood, for thetreatments shown in FIG. 27A; FIG. 27C shows a colored immunofluorescentmicrograph of blood clots in microfluidic channels; FIG. 27D and FIG.27E shows scanning electron micrographs (SEM): FIG. 27D shows unclottedblood and FIG. 27E shows clotted blood in the left panel vs. unclottedblood in the right panel.

FIGS. 28A-C show comparative scanning electron micrographs of FIG. 28Acontrol blood; FIG. 28B blood in the presence of soluble collagen andFIG. 28C blood treated with IC (immune complexes).

FIG. 29 shows a schematic drawing of one embodiment of an exemplarysix-channel microfluidic device where each end of a microchannelterminates at a port. In some embodiments, the inlet port is a largeopening, such as shown on the right. In some embodiments, the largeopening serves as an “on-chip” fluid reservoir (or connects to areservoir). In some embodiments, HUVEC cells coat at least a portion ofthe inside of a microchannel. In some embodiments, HUVEC cells coat theentire microchannel. In some preferred embodiments, the outlet port islocated at the opposite end from the inlet port.

FIGS. 30A-C shows schematic drawings of an exemplary four channelmicrofluidic device illustrating four exemplary embodiments of presetmicrochannel geometries contemplated for use in recreating specificfluidic dynamics of the blood flow. FIG. 30A shows one embodiment of aschematic top view of a four channel chip having four exemplary presetmicrochannel geometries with the same Outflow rate, e.g. having a 100 μmOutflow, also shown in FIG. 30B and FIG. 30C (bottom view), FIG. 30Bshows a schematic bottom view diagram of an exemplary 4 channelmicrofluidic device. FIG. 30C shows one embodiment of a schematic 3-Dangular view of a 4 channel microfluidic device contemplated for use asa mold for fabricating chips shown in FIG. 30A and FIG. 30B.

FIGS. 31A-B show exemplary schematic diagrams of Tile Areas andquadrants representing fields of view, e.g. one embodiment for analyzingevents in microfluidic channels, showing fields of view (FOV) asobserved when viewed through an optical system including but not limitedto an Olympus Light Microscope. The field of view is determined by thefield captured by the camera or the ocular. FIG. 31A shows an outlinerepresenting one field of view (FOV) on a microscope stage, when viewedusing a 10×ocular, e.g. a 1350 μm by 1350 μm area, i.e. one Tile Area asviewed with an Olympus Light Microscope. FIG. 31B shows an exemplaryrepresentation of the total viewing area (e.g. as determined by therange of motion of the stage controls) where the total viewing areaincludes but is not limited to 12 Tile Areas, e.g. 12 quadrants, for atotal view area 5.4 mm wide and 4.05 mm in length, wherein each TileArea or quadrant representing one FOV. In one embodiment, twelve (12)tiles are contemplated for viewing in under a 30 sec frame rate limit (1frame every 30 seconds) for photography, including but not limited tovideophotography, of events occurring within at least one active regionin a microchannel.

FIGS. 32A-C show exemplary schematic diagrams of one embodiment of amicrofluidic chip having 4 channels on one chip. FIG. 32A shows anexemplary 3D view of one embodiment of a microfluidic device, whereinfour lower indentations represent molded on chip reservoirs. Uppercircles represent exemplary inlet/outlet ports. FIG. 32B shows the fourchannels having exemplary dimensions of 100 μm diameter channels spaced50 μm apart for providing a total 550 μm wide region such that parallellocations within the active regions in all four channels may be viewedunder a microscope within a microscopic field of view. In oneembodiment, so that the channels fit under one field, one can designthem as 100 μm×100 μm. FIG. 32C shows an exemplary microfluidic devicedimension of 46 mm long and 18 mm wide, and the dotted lines representthe parallel active regions in the channels as shown in FIG. 32B.

FIG. 33 shows an exemplary schematic diagram of features in oneembodiment of a 4 channel chip. The numbers 1, 2, 3 and 4 (numbers nextto dots) represent inlet/outlet ports for attaching to other components,including but not limited to tubing, e.g. for adding or removing fluids,pump(s) or other devices for inducing negative or positive pressure.Each port may be attached to separate or shared channel control(s), asdescribed herein. Multi-inlets (represented by 2 lower dots) are locatedopposite the EDTA inputs (2 upper dots). In one embodiment, multi-inletsrefer to inlets for cell seeding, cell rinsing, e.g using bufferedsolutions, media, common liquids used in cell culturing, and the like.It is not meant to limit the multi-inlets to inflowing fluids. In someembodiments, Multi-inlets are used for collecting outflowing fluids,e.g. cell media, samples, etc. In one exemplary embodiment, gravitydriven flow for anticoagulants, e.g. EDTA solutions, added into the EDTAinput, provides pressure (gravity) forces for pulsing EDTA into singleadditive channels below, in order to eliminate blood clotting in outflowfrom inlet/outlet ports for collecting and analyzing samples into thereservoirs. Thus in one embodiment, outflow to the reservoirs iscollected from one or more inlet/outlet ports. On chip reservoirs areshown as blocked in the lower part of the drawing. As one example forproviding pressure forces for pushing sample from the reservoirs throughchannels into the upper portion of the device, height of the reservoirsprovides such pressure. For one example, four reservoirs are shown, eachhaving >70 mm{circumflex over ( )}2 area. In one embodiment, a PDMSdevice height is 15 mm with a volume >1 mL. Black lines representbranches and microchannels, black dots represent locations where 2branches microchannels merge into one, or where the microchannelsconnect to a reservoir, in one embodiment, one reservoir is connected toeach (one) channel. It is not intended that the use of multi-inletsapply just to seeding ports and output ports, but also to theanticoagulant ports (EDT), calcium ports, and sample/blood ports.

FIG. 34 shows an exemplary schematic diagram of one embodiment of amicrofluidic chip device as 16 total channels on one chip device. Thearrow points to a region containing sample input ports spaced for usewith a multichannel pipettor, i.e. each port corresponds to thedispensing end of the pipette tip, for simultaneously adding samples ineach input port. One exemplary embodiment shows microchannels 15.6 mm inlength. A chip device size comparison is shown at the bottom with aregular sized American credit card on the left compared to the chipdevice outline shown on the right.

FIGS. 35A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip device related to methods of use. FIG. 35A shows anexemplary schematic diagram of a device during cell seeding, wherepositive pressure, shown by the thick arrows pointing down representingthe direction of fluid flow, is used to seed cells into channels, wherecells are seeded into the multi-inlets while the other ports, 1, 2, 3, 4and EDTA input are plugged (black circles), followed by cell attachmentto the microchannels. Afterwards, medium is pushed through to rinsechannels, see arrowheads in channels/branches between ports and themicrochannels. FIG. 35B shows an exemplary schematic diagram of fluidflow in a device during cell feeding. Medium is added to reservoirs,using 200 ul pipette tips filled with medium inside multi-inlets, whichadditionally serve as plugs during feeding. Pressure used to push mediummay be positive pressure represented by the arrow pointing down, inother embodiments the pressure is negative pressure represented by thearrow pointing up. FIG. 35C shows an exemplary schematic diagram offluid flow in a device during chip prep, where 1, 2, 3, and 4 numberedports are unplugged, while EDTA inlets and multi-inlet ports areplugged. Negative pressure (see direction upwards of thick arrows) isused to fill empty upper channels, then multi-inlets are also plugged.After filling, tubing is attached to inlets 1, 2, 3, and 4 of which atleast one tube is attached to a pump.

FIGS. 36A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip during blood testing. FIG. 36A shows an exemplarydiagram showing where blood is added to reservoirs along with any testagents. Thick arrows show the direction of fluid flow of blood out ofthe reservoirs, with smaller arrowheads showing the direction of flowupwards towards the inlets. FIG. 36B shows an exemplary diagram wherethe four open dots, shown diagonally within the open rectangle (arrow),represent the open (dispensing) ends of pipette tips where the other tipend is attached to a multi-pipetter so that fluid containing an agent,such as a conditional agent, e.g. a coagulation reagent in solution,such as Ca++, intended for adding to blood entering the test channels,is simultaneously added to three ports located below the three lowerdots, one port each for three of the four reservoirs shown as blackareas in the lower part of the chip, where each of the fourmicrochannels is in fluidic communication with a correspondingreservoir. Thus, the solution is mixed into the blood contained in threereservoirs at one time. The remaining reservoir, when receiving asolution as a separate addition into the fourth reservoir port, not inline with the multi-channel pipette tips, upper right, is added/mixedseparately from the other three reservoirs. In some embodiments, thisfourth reservoir is used as a control without the addition of an agentin solution, such as a conditioning solution. FIG. 36C shows anexemplary diagram for preparing Outflow fluid for collection. UnplugEDTA input ports (dots at the top of the diagram), insert the dispensingend of 1 mL syringes for adding EDTA solution. Since a small amount ofEDTA needed, flow downward is gravity driven, see arrowhead pointingdown from the input. Each cm of liquid height=0.1 kPa in pressure; sothat an optimal height of the oar-chip device components is calculatedfor each type of chip.

FIG. 37 shows an exemplary schematic diagram of one embodiment of amicrofluidic chip as a four channel device. In this embodiment, a singleinput port (shown by the circle at the top of the diagram-upperarrowhead) is connected to four branching channels flowing throughresistors (located in between the components identified by the upper twoarrowheads) for regulating the fluidic flow rated for providing equalflow rates of fluids entering each of the four microchannels. Dotslocated after the resistors (middle arrowhead) represents each inlet permicrochannel. The center black lines (as shown in detail in FIG. 31B),represents a side by side viewing area, in part for use in analyzing(including but limited to observing) events occurring in parallel activeregions of the microchannels. The lower blocked rectangles (lowerarrowhead) represents on-chip reservoirs where one reservoir is influidic communication with one of the microchannels. In a preferredembodiment, the dot at the very top is a common calcium port and thefour circles are the blood inlets. This allows the calcium reagent to bethe same across different test conditions, while the different sampleinlets allow the testing of blood from different patients, or blood fromsame patient pre-mixed with different concentrations of a drug. The fourresistors make sure that the same flow rate of calcium goes to all fourtest conditions.

FIG. 38 shows an exemplary schematic diagram of one embodiment of amicrofluidic chip as an eight channel device. In some embodiments, adevice induces, but is not limited to, two eight channel units, wheretwo eight channel units are shown in this figure. In this embodiment, asingle input port (shown by the circle at the top of the diagram—upperarrowhead) is connected to eight branching channels (which in someembodiments are tubes) flowing through resistors (located in between thecomponents identified by the upper two arrowheads) for regulating thefluidic flow rated for providing equal flow rates of fluids enteringeach of the eight microchannels. Dots located after the resistors(middle arrowhead) represent each inlet per microchannel. The centerdark lines (arrow) represents a side by side viewing area (shown in FIG.31B), in part for use in analyzing (including but limited to observing)events occurring in parallel active regions of the microchannels. Thelower shaded rectangles (lower arrowhead) represent on-chip reservoirswhere one reservoir is in fluidic communication with one of themicrochannels. This illustrates how some constructs fall within the samemicroscope field, while others fall within the range of the microscopestage.

FIG. 39 shows an exemplary schematic diagram of one embodiment of amicrofluidic chip device having a single pressure source, i.e. commonpressure source, shown at the top of the diagram, for applying pressureto both an input reservoir (e.g. blood inlet) and an additive reservoir,e.g. containing an anticoagulant solution. Serpentine channels serve asa resistors shown inline between the blood inlet and the chip, and thereservoir and the chip, for regulating the fluidic flow rates. Thedotted lines represent an exemplary chip, e.g. thrombosis chip, whilethe circle in the lower right of the chip area represents and additivechannel area as shown in FIG. 12. In some embodiments, fluidicconnectors are tubes attached to on-chip ports represented by blackcircles within the dotted line outline of the chip. While not intendingto limit the invention in any manner, in one embodiment, the resistorscause the two flows to be proportional under the application of the samepressure. This is useful when it is desired that the anticoagulant orcalcium mix with the blood at a specific ratio to be effective. Forexample, in one embodiment, it is desired that the EDTA finalconcentration be 10 mM, whereas the calcium concentration goes to 20 mM.While FIG. 39 shows the blood inlet and anti-coagulant in separatecontainers, the present invention also contemplates the situation wherethe pressure source acts on a single container or component (e.g. areservoir) that has been divided. For example, in one embodiment, thepresent invention contemplates modifying an existing reservoir into tworeservoirs with a dividing wall.

FIGS. 40A-C shows an exemplary photographs of both types of reservoirs,external (as an exemplary syringe) and internal (as on chip), at theblood inlet of a microfluidic chip. FIG. 40A shows an exemplary offcenter overhead view of a reservoir assembly on a microfluidic chipwhere an arrowhead points to the connection between the tiler of asyringe (representing an off-chip reservoir) with a blood inlet foradding fluid to an on chip reservoir. A white arrow points to anexemplary on-chip reservoir. FIG. 40B shows an exemplary overhead viewof syringe attached to chip a reservoir as an assembly in the bloodinlet. A circular component on the right of the chip represents a port.FIG. 40C shows an exemplary chip showing an enlarged view of theconnection between the off chip (syringe) and on chip reservoir (arrow)at a blood inlet, showing the luer connection (arrowhead) with the inletport.

FIGS. 41A-D shows exemplary schematic drawings of one embodiment of amicrofluidic chip device demonstrating additional details of some of thecomponents as shown in FIGS. 32A-C and 33. FIG. 41A shows an enlargedillustration of the branches (short arrows) merging with channels (longarrows) as shown within the circle labeled A in FIG. 41C. The arrowheadpoints to an exemplary input between a branch and a channel. FIG. 41Bshows an enlarged illustration of the channels shown within the circlelabeled B in FIG. 41C. FIG. 41C shows an illustration of themicrofluidic device. FIG. 41D shows a 3D illustration of themicrofluidic device where the branches (short arrows) and channels (longarrows) are shown in the black area and on-chip reservoirs (opentriangles). The double headed arrow points to 2D vs. 3D drawings ofcorresponding reservoirs between FIG. 41C and FIG. 41D, respectively.

FIGS. 42A-D shows exemplary schematic drawings of one embodiment of amicrofluidic chip device demonstrating additional details of some of thecomponents as shown in FIGS. 37 and 38. FIG. 42A shows an enlargedillustration of the channels in the active regions as shown within thecircle labeled A in FIG. 42C. FIG. 42B shows an enlarged illustration ofa resistor region shown within the circle in FIG. 42C. FIG. 42C shows anillustration of one embodiment of a microfluidic device where a singleport has multiple branches, where each branch has a resistor, shownwithin the circle, such that after fluid flows through the resistorseach branch has a flow rate equal to the other branches flow rate as thefluid enters the channels. FIG. 41D shows a 3D illustration of oneembodiment of a microfluidic device where the branches (short arrows)and channels (long arrows) are shown in the black area. The doubleheaded arrow points to 2D vs. 3D drawings of corresponding reservoirsbetween FIG. 42C and FIG. 42D, respectively. In some embodiments, theeight circles at the bottom of each of the two chip units shown (8 perchip) represent the output. In some embodiments, there is a singleadditive channel input (see the single circle at the bottom of the chipunit diagram that feeds in through a resistor (i.e. switchbacks orsquiggles) that is associated with each of the 8 outputs.

DESCRIPTION OF THE INVENTION

The present invention contemplates compositions, devices and methods ofpreventing, reducing, controlling or delaying adhesion, adsorption,surface-mediated clot formation, or coagulation in a microfluidic deviceor chip. In one embodiment, blood (or other fluid with blood components)that contains anticoagulant is introduced into a microfluidic devicecomprising one or more additive channels containing one or more reagentsthat will re-activate the native coagulation cascade in the blood thatmakes contact with it “on-chip” before moving into the active orexperimental region of the chip.

I. Advantages of a Microfluidic-Chip Device

Advances in microfluidic engineering have recently made it possible tocreate miniaturized in vitro cell culture systems, known asorgans-on-chips^(29,30), in which human cells and tissues are subjectedto fluid flow and mechanical stress in well-controlled three-dimensionalgeometries as microenvironments. Thus, organs-on-chips producehuman-relevant physiological data that is used in biomedical science,toxicology and pharmacology.

Systems comprising organ components allow for controlled studies oforgan-level aspects of human physiology and disease, and weresuccessfully applied in the preclinical testing of therapeutics³¹.Microengineered on-chip systems containing human endothelium, perfusedwith human whole blood at physiological relevant shear rates,recapitulate many aspects of thrombosis (Jain, et al. Assessment ofwhole blood thrombosis in a microfluidic device lined by fixed humanendothelium. Biomedical Microdevices. 18:73, 2016; Westein, et al.“Atherosclerotic geometries exacerbate pathological thrombus formationpoststenosis in a von Willebrand factor-dependent manner,” Proc. Natl.Acad. Sci. U.S.A. 110, 1357-1362 (2013); Tsai, et al. In vitro modelingof the microvascular occlusion and thrombosis that occur in hematologicdiseases using microfluidic technology. J. Clin. Invest. 122, 408-418(2012); Westein, et al., “Monitoring in vitro thrombus formation withnovel microfluidic devices.” Platelets 23, 501-509 (2012). However,these on-chip blood vessels lack human-relevant physiological data.Unlike other systems, such as the Badimon chamber, which are designedfor real-time monitoring for anti-coagulant properties of drugs in aclinical setting, the microengineered Vessel-On-Chip was designed, atleast in part, for preclinical testing to select candidate drugs forefficacy (e.g. anti-coagulants) or for safety (e.g. risk forthrombosis).

Therefore, a microfluidic-chip device was developed, as describedherein, for overcoming such limitations of other chips for providinghuman-relevant physiological data. In one embodiment, aThrombosis-On-Chip was created in part by perfusing microfluidic chipswith human blood with au anticoagulant, such as citrate solution, addedto outflowing blood samples to prevent clotting inside connectors andtubing. In some embodiments, a Vessel-On-Chip was created which in partprovides advantages in allowing analysis of the outflowing blood samplesover longer durations in time of experimental testing. In someembodiments, a Vessel-On-Chip was created which in part providesembodiments for testing certain combinations of healthy components,diseased components, and some healthy components with some diseasedcomponents. Components include but are not limited to normal healthyblood components, normal healthy cells, treated blood, treated cells,blood from diseased patients, cells from diseased patients, etc.

Advantages of using a microfluidic chip device (alternatively, amicrofluidic chip) as described herein, include but are not limited to:providing a system enabling control over blood parameters, e.g. changingflow rates, changing components interacting within blood, etc.;providing a model designed to interface with microscope, e.g. enablinghigh speed real time imaging, etc., and providing an analysis on smallvolumes of fluids, e.g. using minimal volumes of blood. Further,advantages of using a microfluidic device as described herein, overother systems include but are not limited to: Capability to perform longterm experiments; blood vessel-endothelial cell interaction, for oneexample, when cells line the device channels so there is not directinteraction between blood and device material interaction; directmonitoring and record of the inflammatory response; direct monitoringand record of blood reactivity; providing multiple modular geometrieseach modeling a specific aspect of flow dynamics; and potentialapplication for patient-specific modeling the blood reactivity in nitro,e.g. for use in personalized medicine.

A. Embodiments of Microfluidic Chip Devices.

In some aspects, materials and methods are provided herein for use withmodeling of blood flow and its effect on cells. In other aspects,materials and methods are provided herein for modeling the effect ofcells on blood flow and its properties. Additionally, many variations ofthe materials are contemplated for use in providing microfluidicdevices, including but not limited to materials allowing for partial orfull views of microfluidic channels.

Thus in one embodiment, modeling blood flow on a microfluidic chipmimics events associated with blood clotting, such asThrombosis-On-Chip. In some embodiments, modeling blood flow on amicrofluidic chip involves lining microfluidic channels with endothelialcells.

In one embodiment, modeling blood flow on a microfluidic chip enableshematodynamic modeling, including but not limited to cardiovascularsystem (involving wave propagation and flow-induced instabilities ofblood vessels, capillary-elastic instabilities, etc.

In some embodiments, modeling blood flow on a microfluidic chip involveslining microfluidic channels with cells derived from blood vessel cells,for e.g. cells isolated from blood vessels obtained from patients(including but not limited to live patients undergoing biopsies,surgery, etc), from cadavers and from commercial sources. In someembodiments, modeling blood flow on a microfluidic chip involvesspecific geometries for producing certain types of biofluid mechanicswithin tubes, e.g. including but not limited to rigid microchannels, inpart for identifying factors related to fluid mixing, internal flow,effects on blood clotting, etc.; flexible microchannels, in part foridentifying factors when mimicking vessel wall deformation in relationto internal flow, effects on blood clotting, pressure-drop/flow-raterelations, and combinations thereof.

Cells may refer to cells disposed within or coating a microchannel, suchas endothelial cells, and cells contained in the blood added to themicrochannels. When we mention cells contained into the blood we referboth to cells normally present in the blood of healthy patient (whitecell, red cell and other particulates such as platelet) and cells notnormally present into the bloodstream such as metastasis or othermicroorganisms which could be present in diseased patient.

Contemplated experiments focus on several aspects, such as measuringhow: 1) mechanical forces and geometry of blood vessel affect thebehavior of the cells contained into the blood; 2) blood cells alter therheological properties of blood in reaction to mechanical stresses; 3)blood/endothelial cells interaction affects the theological propertiesof the blood and the properties of the endothelia surface; 4) epithelialand parenchymal cells affect endothelial cells and rheological bloodproperties in response to external stimuli and stresses (chemical,mechanical, biological etc), and the like.

In other words microfluidic devices (including chips) described hereinare contemplated to provide a means for: 1) recreating severalperfusable vessel geometries which can mimic an actual healthysimulation using components from healthy patients, mimic a diseasedblood vessel using components from patients with a disease or at risk ofa disease, and mixed simulations using combinations of components fromhealthy patients with components from patients with a disease or at riskof a disease; 2) lining the vessel with living endothelial cellsspecific for (i.e. isolated from or derived from or associated with)each specific organ under study; 3) creating vessels for use in flowingblood through (i.e. through a microchannel lined with simulated bloodvessels), where blood is isolated from an individual patient; 4)providing a system for integration with the Organ-on-Chip concept andused to study the effect of the interaction of multiple cell types onblood (for example, one can study how epithelial/parenchyma/mesenchymalcell interact with endothelial cell and how those affect the blood andvice versa); 5) visualizing the lumen of reconstructed blood vessels(i.e. blood vessel mimics) using microscopy at high speedviewing/imaging/recording such as with photography/videos and highresolution (i.e. capable of subcellular level viewing).

B. Computational Hemodynamic Modeling.

Computational hemodynamic modeling is divided into at least three maindescriptive groups:

1) A real time imaging system refers to imaging based on dopplerultrasound, CT (Computed tomography) angiography and MR (magneticresonance) angiography, or similar which produce image or video of theblood flow in real time within a patient. Thus, real time imagingsystems refer to devices capable to visualize the lumen of blood vesselsin the body. However, in general, application of this techniques arelimited because their use requires exposure to x-ray or radioactivecontrast agents which limit their application on human for safetyreason. Long term exposure to such agents increase exponentially therisk to develop a cancer, plus high doses of radiation commonlyassociated with various side effects which include nausea, vomiting,pain, swelling, redness or cutaneous rash of the treated area. Othertechniques based on the use of ultrasound are generally safer, but theprolonged use on patients is not recommended or in some casescontraindicated. Another limiting factor in the use of these techniquesfor modeling blood resides in the fact that these techniques have alimited resolution (generally sub millimeter), which does not allow tovisualize cellular structure or to perform studies involving the effectof the blood flow on cells.

2) Computational modeling may also provide a mathematical description ofblood flow, in models were the blood is approximated to an inertcolloid/viscoelastic fluid. This type of modeling generally requirescomputation analysis. Application of computational modeling is generallylimited in biology because such models merely provide a mathematicaldescription of the blood as a fluid, which does not include effects ofthe behavior of cells within the blood and further does not provide adescriptive model of the behavior of cells contained within blood andtheir effect on blood properties or to describe blood interaction withother cells. Since computational models are incapable of modeling cellbehavior they are intrinsically inaccurate to describe the biology ofblood.

Therefore, at least in part to address unmet needs, we have developed anoptically transparent platform made of PDMS for hemodynamic modeling andtesting, which is a tools for modeling the physic of blood flow and thebiological behavior of blood cells in it contained under preset fluidicdynamic regimes and that allows 3D imaging at high resolution in realtime of the blood flow. The specific setup can also be used asdiagnostic tools in personalized medicine.

High resolution high speed computational imaging of the blood enables adetailed visualization of the complex shear stress velocity and thepressure fields which can be directly correlated with cellular behavior.Since the platform is a modular system and the fluidic parameters can befinely tuned the system can be set to extrapolate information atdifferent length scale by connecting several modules containing aspecific geometry either in parallel or in series and time scale bysetting different working time on the peristaltic pump.

Furthermore, the platform could be used as a diagnostic tools inpersonalized medicine application. Specifically, after defining specificmetrics using blood from patients with, blood disorders, it would bepossible to make prediction about their response to medical treatments,drugs and diets. In the future, one can envision the routineincorporation of these data in hospital practice to help virtualtreatment planning of the patient as it occurs already in other medicaldisciplines.

This modular platform permits to integrate multipletheological/geometrical units into a comprehensive system to investigatethe impact of various conditions simultaneously. The full-integratedsystem offers the possibility of understanding, holistically, the impactof cardiovascular disease upon individual patients.

The microfluidic cip devices described herein, are contemplated for usewhen integrated with other types of microfluidic chip devices and usedto study the effect of the interaction of multiple cell types on blood,to study how inflammation of epithelia and parenchyma, drugs, chemicalcompounds and physical forces affect/influence the behavior of theendothelial cells lined in the channel and how all of them can affectblood and blood-cells behavior (white cells, red cells and platelets).The applications of the system involves also, but is not limited, to thestudy of complex events such as thrombosis, thromboembolism, aneurism,atherosclerosis, ischemia and the significance of lesions generated bypressure and other mechanical stresses that can affect blood andendothelial cells.

C. Microfluidic Devices for Studying Thrombotic, Blood Clotting, Events

In addition to evaluating clinically relevant aspects of thrombosis,such as platelet-endothelium interaction, platelet aggregation andfibrin formation, studied in vitro within a microfluidic-chip device,these microfluidic chips can be used to detect early stages indrug-induced thrombosis and thromboembolism.

Further, additional advantages of using microfluidic-chip device testingof thrombosis include but are not limited to evaluating solublebiomarkers. As described herein, we demonstrate the significance of ourmodel for preclinical drug testing in one example, by studying thepro-thrombotic effects of hu5C8. Our results confirm that plateletadhesion to endothelium, platelet aggregation, and fibrin formation canbe measured and visualized in microfluidic-chip device systems. Inaddition to detecting platelet adhesion to the endothelium, theformation of a network of fibrin clots within the chip followingtreatment was confirmed by imaging and scanning electron microscopy.

This microfluidic-chip device design enabled collection of eluents, froman additive channel attached to the outflow port, for quantification ofbiomarkers such as the thrombin-antithrombin complex (TAT).

Another advantage of using a microfluidic-chip device was demonstratedby its use to analyze multiple aspects of thrombosis, such as plateletadhesion, aggregation, fibrin formation and TAT release, all in a singleassay.

A microfluidic-chip device is contemplated for use in testingpatient-specific blood as it flows through the chip, including but notlimited to patient-specific normal and/or diseased tissue/cells.

Thus drug treatments, e.g. anticoagulants, such as an anti-plateletdrug, including but not limited to FDA-approved Eptifibatide, may betested in vitro. Further, candidate therapeutic drugs, such astherapeutic antibodies, e.g. treatment using hu5c8 as an immune complexwith sCD40L: IC_(5c8), may be used to identify potentially adverse sideeffects as shown herein, that duplicated adverse side found during humanclinical trials.

In some embodiments, the resulting characteristic temporal and spatialindices were sensitive enough to distinguish activated platelets (e.g.,due to inflamed endothelial cells) and non-activated platelets. Thus,the temporal and spatial indices can be used as markers to diagnosediseases or disorders (e.g., platelet-associated disease or disorder),to select appropriate therapy (e.g., anti-platelet and/oranti-inflammation therapy), to monitor treatment efficacy (e.g., toprevent recurrent thrombosis or bleeding), drug screening and/or todetermine drug toxicology. Accordingly, embodiments of various aspectsdescribed herein relate to methods, systems, and compositions fordetermining dynamic interaction of cells with each other, and/or withother cell types, and uses thereof.

Moreover, a microfluidic-chip device is contemplated for use inidentifying “at risk” patients, e.g. so that simulated clinical trialsmight be done using blood and cells from a patient in vitro, thusreducing actual harm in patients that may have an adverse reaction to aparticular therapy if tested in vivo.

In some embodiments, the present invention contemplates a method ofdetermining if a subject is at risk, or has a disease or disorderinduced by cell dysfunction and/or abnormal cell-cell interaction. Themicrofluidic device can be used for diagnosis and/or prognosis of adisease or disorder induced by blood cell dysfunction (e.g., plateletdysfunction), and/or guiding and/or monitoring of an anti-plateletand/or anti-inflammation therapy. Non-limiting examples of the diseaseor disorder induced by blood cell dysfunction (e.g., plateletdysfunction) include, but are not limited to thrombosis, an inflammatoryvascular disease (e.g., sepsis, or rheumatoid arthritis), acardiovascular disorder (e.g., acute coronary syndromes, stroke, ordiabetes mellitus), vasculopathies (e.g., malaria, disseminatedintravascular coagulation), or a combination of two or more thereof.

In one embodiment, the pro-thrombotic effects of drugs and antibodiesare revealed by in vitro testing in the microfluidic device. In oneembodiment, the pro-thrombotic effect of hu5C8 was revealed in vitrousing disease-relevant concentrations of sCD40L and clinically relevantconcentrations of hu5C8. Indeed, previous studies were conducted usingplatelets frequently exposed to supraphysiological concentrations ofsCD40L (1000 times higher than serum levels of sCD40L found in diseasestates)^(23,27). In our model, thrombosis induced by hu5C8 was dependenton the FcγRIIa receptor. In fact, thrombosis was prevented by IV.3, ablocking antibody against FcγRIIa and hu5C8-mediated thrombosis was notdetected when we used hu5C8-IgG2σ, a molecule formatted not to bind theFcγRIIa receptor. Our results provide confidence that the newergeneration anti-CD154 mAbs that do not bind FcγRIIa receptors have a lowrisk for thrombosis²³. The ability of this microfluidic Vessel-On-Chipto provide reliable measurements of clinically relevant endpoints makesit a suitable platform to assess risk for thrombosis of a broad class ofmolecules developed for therapeutic applications.

II. Incidents of Drug-Induced Thromboembolism During Clinical Trials.

Activation of T cells via CD40 ligand (CD40L/CD154) binding is one steptowards the initiation of the adaptive immune response. Blocking ofCD40L-mediated signaling represents a powerful therapeutic strategy¹ fortreatment of auto immune disorders² and for preventing organ transplantrejection³. Pre-clinical studies conducted on animal models havedemonstrated that monoclonal antibodies (mAbs) against CD40L can be usedto suppress organ transplant rejection or the auto-immune response.However, the development of anti-CD40L mAbs was halted for several yearsbecause of multiple incidents of thromboembolism and cardiovascularevents during clinical trials of the drug candidates hu5C8 and IDEC-131,which were under development for treatment of lupus^(9,10) and/orCrohn's disease^(11,12,13-15).

In addition to expression on T cells, CD40L is also expressed onactivated platelets where, after translocation to the surface, it shedsas soluble CD40L (sCD40L)¹⁶. Platelets also represent the major sourceof circulating sCD40L⁷⁻¹⁹, with high concentrations reported in patientswith inflammatory diseases^(20,21). It is believed that thrombosis byhu5C8 is mediated by ligation of a high-ordered immune complex (IC) ofhu5C8 with sCD40L to the FcγRIIa receptor specifically expressed onhuman platelets^(10,22-27). Given these findings, new anti-CD40L mAbsunder development should have both a demonstrated efficacy along with agreat degree of confidence (i.e. data supporting) a lack thrombosisinduction in the presence of sCD40L²⁸. The lack of robust models able topredict the mAb-mediated thrombosis by this complex mechanism is one ofthe main obstacles for the advancement of new monoclonal therapeuticsagainst CD40L. While platelet aggregation and activation continue to bethe gold-standard for in vitro assessment of risk for thrombosis, an invitro model that truly captures the complexity of human plateletaggregation as well as activation of the coagulation cascade wouldaddress the unmet need for systems that can aid in selecting compoundswith reduced risk for thrombosis.

As described herein, we demonstrate that Vessel-On-Chip technology issuitable for studying drug-induced thrombosis that is relevant to humansin the context of disease and drug treatment. Perfusion of a biomimeticvessel on-chip with human blood samples containing pathophysiologicallyrealistic levels of the inflammatory cytokine sCD40L (CD154) andrelevant concentrations of hu5C8, an anti-CD154 monoclonal antibody thatwas intended for treatment of autoimmune disorders, leads to enhancedplatelet-endothelial adhesion, platelet aggregation, and fibrin clotformation. The thrombotic endpoints detected in the chip are consistentwith clinical findings of thrombosis by hu5C8 that led to termination ofits clinical trials. In addition, treatment-related increase in thrombinanti-thrombin (TAT) complex in the eluate from the chips demonstrates anability to couple imaging endpoints with biomarker detection in thismodel. The thrombotic effects could be prevented by a blocking antibodyagainst FcγRIIa receptors expressed on platelets or by using ananti-CD154 mAb modified not to bind FcγRIIa receptors. Our resultsdemonstrate that Vessel-On-Chip technology can be used to detectclinically relevant thrombotic effects in vitro, even when such effectsare mechanistically complex.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

FIG. 1 schematically shows one embodiment of a microfluidic device 200that includes a plurality of ports 205 in fluidic communication with amicrochannel 203. The center of the microchannel 203 serves as theactive region or experimental region (see 207 of FIG. 1 and FIG. 2). Theactive region or experimental region is typically where cells arecultured, where cells interact, and/or where cells are tested. In oneembodiment, the active region is a tissue-tissue interface simulationregion where cell behavior and/or passage of gases, chemicals,molecules, particulates and cells are monitored. This region can bemonitored, e.g. with a microscope or other imaging system (not shown).Also shown is one embodiment for the outer body 202 of the device 200;input port 211 and output port 215; optional vacuum chambers 252 and ahorizontal orientation plane in dotted lines A, including an exemplarycross-section of microchannel 203.

FIG. 2 illustrates an exploded view of the embodiment shown in FIG. 1.In particular, the outer body 202 of the device 200 is preferablycomprised of a first outer body portion 204, a second outer body portion206 and an intermediary porous membrane 208 configured to be mountedbetween the first and second outer body portions 204, 206 when theportions 204, 206 are mounted to one another to form the overall body.Both input ports 211; 219 and output ports 215; 221 with contact regions210, 212, 218 and 220, respectively; inflow channels 225 and outflowchannels 227; microchannels 203 with a center region 207; in addition tosupplementary ports 217, 223, 227 and 229, with corresponding contactregions 214, 215, 222 and 224; are shown. Further, membrane 208 has twosurfaces 208A and 208B. Dotted lines show horizontal orientation planesC and D, including exemplary cross-sections of microchannels 203.

In one embodiment, the present invention contemplates the inclusion ofadditional fluid inputs to increase the mixing of reagents and cells,and in particular, the mixing of anticoagulant with blood. FIGS. 3A-Cshows one embodiment of on-chip mixing using a single input, i.e. asingle additive channel in fluidic communication. FIG. 3A shows theadditive channel attached to the microfluidic port of the microfluidicdevice. FIGS. 3B and 3C are photographs showing the single input streamand single input mixing. FIGS. 4A-C show one embodiment of on-chipmixing using a dual input, i.e. two additive channels, one on one sideof the microchannel (near the input port) and on the other side of themicrochannel. FIG. 3A shows a schematic of the two additive (dual input)channels attached to the microchannel of the microfluidic device (nearthe input port of the microfluidic device). FIG. 4B and FIG. 4C arephotographs showing the dual input streams and dual input mixing,respectively. Both a single input and double input additive channelsminimize the exposure of activated blood to components of the chip, i.e.exposure to materials that may impact the results in the active regionor experimental region of the chip.

One embodiment of on-chip mixing uses a dual input, i.e. two additivechannels in fluidic communication is shown in FIGS. 4A-C. FIG. 4A showsa schematic of the dual input additive channels attached to themicrofluidic port of the microfluidic device. FIG. 4B and FIG. 4C arephotographs showing the dual input streams and dual input mixing,respectively.

FIGS. 5A-B shows exemplary photographs demonstrating on-chip mixing ofanticoagulant. FIG. 5A shows tubing connecting a source of sodiumcitrate to the microfluidic device (not shown). On-chip mixing withsodium citrate (see tag—pointed at by the arrow) allows samples to flowfreely, while lack of anticoagulant input clogs collection tubes and canslow or completely stop flow. FIG. 5B shows five tubes, four of whichwere treated on-chip with anticoagulant and can be analyzed. Tube 5 wasnot treated and contains a solid mass of coagulated blood, which cannotbe used for testing, FIG. 5B shows upside down microcentrifuge tubeswith blood clots in the pointed ends and free blood cells in solution inthe lower flat ends. These tubes started upright (pointed ends down),with 1-5 different treatments of a liquid solution of red blood cells.Then the tubes were spun down in a microcentrifuge on an angle with thepointed ends down. After the tubes were taken out of the centrifuge theywere placed upside down with the clots staying in the pointed ends, withsmaller—lighter clots sticking to the tube along one side (because itwas on an angle as it was spun (tubes 1-4)) and free red blood cellsthat fell by gravity through the clear fluid into the lower ends wherethe numbers are located. Tube 4 has more free red blood cells than Tubes1-3. Unlike Tubes 1-4, Tube 5 has heavy blood clots that stayed in thepointed end with clear fluid below.

In one embodiment, blood (or other fluid with blood components) thatcontains anticoagulant is introduced into a microfluidic devicecomprising one or more additive channels containing one or more reagentsthat will re-activate the native coagulation cascade in the blood thatmakes contact with it “on-chip” before moving into the experimentalregion of the chip. FIGS. 6B and 6C are images showingfluorescently-labeled platelet aggregates, demonstrating clotting in theuntreated sample. FIGS. 6D and 6E are images for the treated sample,with labeled platelets from the anticoagulant treated sample that aredispersed in the blood liquid phase, demonstrating an uncoagulatedstate. The platelets were labeled with CD41-TRITC antibody,demonstrating that Cells can be visualized with antibody binding.

Blood contains many cell types along with the plasma liquid phase, allof which are available for study and testing in uncoagulated samples. Avariety of tests can be performed in the microfluidic device usinganticoagulated blood, including but not limited to platelet functiontests, including clot measurement. FIG. 7A shows the size distributionof clots in samples from various donors. FIG. 7B shows that the analysisof soluble factors (TAT) in response to the addition of procoagulantfactors to the blood.

The additive channel(s) or the microchannel itself can contain featuresthat increase mixing. For example, in one embodiment, the additivechannel(s) or microchannel (or portion thereof) can contain ridges. Inone embodiment, the present invention contemplates a three-dimensionaltwisting flow in a mixing microchannel channel with ridges on one wall(or a portion of one wall, e.g. at the beginning before the activeregion). FIG. 8 shows a schematic diagram of microchannel with obliquelyoriented ridges for enhanced mixing. The coordinate system (x y z)defines the principal axes of the channel and of the ridges. The angle(theta) defines the orientation of the ridges with respect to thechannel. The amplitude of the ridges is small compared to the averageheight of the channel. The width of the channel is w and principalwavevector of the ridges is q. The lines represent trajectories in theflow. The streamlines of the flow in the cross section are shown belowthe channel. The angular displacement is evaluated on an outerstreamline.

FIG. 9 shows one embodiment of a microchannel (or portion thereof) witha staggered herringbone (SH) design of ridges for enhanced mixing. Theschematic diagram shows one-and-a-half cycles of the SH. In oneembodiment, a mixing cycle is composed of two sequential regions ofridges; the direction of asymmetry of the herringbones switches withrespect to the centerline of the channel from one region to the next.The streamlines of the flow in the cross section are shown schematicallyabove the channel. The average angular displacement of a volume of fluidalong an outer streamline over one half cycle in the flow generated bythe wide arms of the herringbones can be calculated. The fraction of thewidth of the channel occupied by the wide arms of the herringbones is p.The horizontal positions of the centers of rotation, the upwellings, andthe downwellings of the cellular flows are indicated by c, u, and d,respectively.

FIGS. 10A-D shows schematics depicting platelet thrombus formation overa monolayer of living endothelium. In a microchannel covered on allsides with untreated living endothelium (FIG. 10A), whole blood flowswithout clotting (FIG. 10B). In contrast, platelet-rich thrombus forms(FIG. 10C) if the endothelium is prestimulated by a pro-inflammatorycytokine, such as TNF-alpha, due to expression of procoagulatoryproteins at its surface (FIG. 10D). In some embodiments, the responsesof blood under flow shown in the figures can be reconstituted usingsimilar microchannels that are lined by a chemically preserved (e.g.fixed) endothelium.

FIG. 11 is a diagram of a simple microfluidic device or chip showinginlet and outlet ports in fluidic communication with a singlemicrochannel, with the active region or experimental region of thechannel highlighted (dashed line).

In one embodiment, the blood (or other fluid with blood components) isfurther treated as it leaves the active region of the microchannel, orimmediately thereafter, in order to reduce the chance of clotting aftertesting. In one embodiment, the present invention contemplates one ormore additive channels (positioned near an output port) containing oneor more reagents that will inactivate the native coagulation cascade inthe blood that makes contact with it “on-chip” as it leaves the activeor experimental region of the chip, permitting the blood to flow out theoutput port. While one additive channel can be used, it has been foundempirically that two channels (one on either side of the output port orend of the microchannel) better control clotting.

FIG. 12 shows an embodiment with two additive channels are employed. Asthe sample flows towards the outlet (output port), an anticoagulant(e.g. EDTA) is introduced from both sides of the microchannel via thetwo additive channels in fluidic communication therewith. The EDTAenters and coats the sides of the microchannel (FIG. 12, see arrows),reducing the chance that the blood will contact the walls of themicrochannel. As the blood flows (see arrows) the EDTA mixes with thatportion of the blood coming in contact with it. FIG. 12 is a schematicof one embodiment of a microfluidic device showing the end of themicrochannel and outlet port in fluidic communication with two additivechannels comprising anticoagulant, so that the sample leaving themicrochannel remains fluid as it approaches the outlet port and exitsthe microfluidic device. It is not meant that 100% of the EDTA (or otheranticoagulant) coats the sides of the microchannels. Indeed, some of theEDTA mixes with the blood sample flowing through the microchannel (seelarge arrows demonstrating fluid flow from the channel towards theoutlet). In other words, EDTA flowing from the EDTA, input contactsblood flowing through the microchannel (demonstrated by smallerdirectional arrows pointing towards the outlet) then flows as a mixedsolution out of the Outlet port. It is desired that the amount of EDTA(or other agent) be sufficient to prevent the blood from coagulating inthe channel. Thus, a ratio of EDTA to blood is contemplated in someembodiments.

FIG. 13 is a schematic of a partial top view of one embodiment of amicrofluidic device (the complete device is not shown) showing one endof the microchannel 1301 terminating at a first port 1302, themicrochannel in fluidic communication with two additive channels 1303and 1304 (one on either side) connecting to a second port 1305 (e.g. foradding the additive). Thus, in one embodiment, an additive channellocated at a port has a single input port for the additive. In otherwords, one additive channel may have an individual additive input port,not shared with another additive channel. However, it is not meant tolimit the number of additive channels connected to one additive inputport, see exemplary embodiments in FIG. 13, wherein one embodiment shows1305 as a single input port for two fluidically connected additivechannels, 1303 and 1304. Thus, in one embodiment, two “additivechannels” are plumbed together to a single additive input port,sometimes this port is referred to as an “EDTA input”, see FIGS. 34 and35. While the term EDTA input is used herein, it is not meant to limitthe type of anticoagulant introduced into an EDTA input, such that othertypes of anticoagulants such as citrate, etc., may also be added forflowing through an EDTA input as an additive channel.

FIG. 14 is a schematic of a complete top view of one embodiment of amicrofluidic device 1400 showing two microchannels 1401 and 1402 alignedwith one another (i.e. the main body 1403 of the first channel is abovethe main body 1404 of the second channel), where each end of eachmicrochannel terminates at a port 1405 and 1406, wherein the firstmicrochannel is in fluidic communication with two additive channels 1407and 1408 (one on either side just prior to the port), wherein the secondmicrochannel is in fluidic communication with two additive channels 1409and 1410 (one on either side just prior to the port), each additivechannel connecting to separate port 1411 and 1412 (e.g. for adding theadditive).

FIG. 15 is a schematic side view of the embodiment of a microfluidicdevice 1500 shown in FIG. 14, where each of two microchannels 1501 and1502 terminates at first and second ports 1504-1506, wherein the firstmicrochannel is in fluidic communication with two additive channels 1507and 1508 (one on either side just prior to the port), wherein the secondmicrochannel is in fluidic communication with two additive channels 1509and 1510 (either side near the port), each additive channel connectingto a separate port 1511 and 1512 (e.g. for adding the additive), 1503and 1505 are sample inlet ports.

FIG. 16 is a schematic top view of one embodiment of a microfluidicdevice 1600 having four microchannels 1601-1604 in parallel, where eachend of the first microchannel 1601 terminates at first 1605 and secondports 1606, where each end of the second microchannel 1602 terminates atfirst 1607 and second ports 1608, where each end of the thirdmicrochannel 1603 terminates at first 1609 and second ports 1610, whereeach end of the fourth microchannel 1604 terminates at first 1611 andsecond ports 1612, wherein each microchannel is in fluidic communicationwith two additive channels 1613-1620 (one on either side just prior tothe port), each additive channel connecting to separate port 1621-1624(e.g. for adding the additive).

FIG. 17 is a schematic side view of the embodiment of a microfluidicdevice 1700 shown in FIG. 16, where each end of the first microchannel1701 terminates at first 1705 and second ports 1706, where each end ofthe second microchannel 1702 terminates at first 1707 and second ports1708, where each end of the third microchannel 1703 terminates at first1709 and second ports 1710, where each end of the fourth microchannel1704 terminates at first 1711 and second ports 1712, wherein eachmicrochannel is in fluidic communication with two additive channels1713-1720 (one on either side just prior to the port), each additivechannel connecting to separate port 1721-1724 (e.g. for adding theadditive).

Some of the figures above illustrate a version of the chip with additivechannels on one side. This can be the output side or the input side, orboth. Were the additive channels are on the output side, this could beused, for example, for adding anticoagulant (e.g. to permit capturingblood that has gone through the chip for downstream analysis, and alsofor avoiding clot formation in the output, which can lead to cloggingand reduced flow), However, the design can be flipped, with similaradditive channels on the input side. These may be used, for example, toadd calcium to blood (containing anticoagulant) in order render theblood capable of coagulation in preparation for a coagulation-relatedtest or experiment. As one example, see FIG. 39.

Thus, in one embodiment, wherein a microfluidic chip has an upper andlower microchannel, each connected to an inlet port and outlet port, onemicrochannel may have two “additive channels” located at the terminationport (i.e. OUTLET port). However, it is not meant to limit the number ofports (i.e. microchannels) having at least one additive channel. Indeed,in other embodiments, each outlet port, i.e. each microchannel, may havetwo “additive channels” located at each of the termination ports (i.e.OUTLET ports), see exemplary embodiments in FIG. 14 and FIG. 15. In oneembodiment, wherein a microfluidic chip has an upper and lower channel,one or both microchannels may have two “additive channels” located atthe inlet ports. In yet a further embodiment, wherein a microfluidicchip has an upper and lower channel, both channels may have two“additive channels” located at one or both inlet ports and two “additivechannels” located at one or both outlet ports, see, FIG. 39.

The presence of additive channels is not limited to microfluidic deviceshaving upper and lower channels. Thus, any sample inlet port may alsohave additive channels which are in turn connected to one or more inputports. In one embodiment, such additive channels for sample inlets mayenable the addition of a de-anticoagulating reagent, e.g. calcium, inaddition to other agents for testing in blood flowing through amicrofluidic channel towards an outlet port. In yet further embodiments,a microfluidic channel may have additive channels at both the sampleinlet port and sample outlet port.

I. Engineering a Microfluidic-Chip Device.

The design of a biomimetic microfluidic-chip device for “on chip”modeling associated with blood as described herein, was made after usingprevious chamber designs that were found not suitable for perfusion ofhuman blood because, in part, the blood components would immediatelybegin coagulating as they flowed through tubing, channels, etc. Thus, inorder to overcome this limitation, one embodiment of a microfluidic-chipdevice referred to as a Vessel-On-Chip has at least one or more additivechannels that are not part of a previous organ-on-chip designs, asdescribed herein.

Thus, in one embodiment, a microfluidic-chip device comprising twomicrochannels, one upper channel and one lower channel coated withvascular endothelial cells is referred to as a Vessel On-Chip because itwas proven to be suitable for endothelial cell growth and maintainingtissue barrier function. In part, one embodiment of a microfluidic chipdesign described herein, has a modification of a previous lung-on-chipwhere the lung-on-chip has a 400 μm chamber, to maximize the contactarea existing between endothelial cells and perfused human blood. Thus,in one embodiment of a microfluidic-chip device, the width of thechamber is 1 mm, scaled up from the 400 μm chamber of the lung-on-chip(FIG. 1 and FIG. 2: channels 203) in a Vessel-On-Chip.

Briefly, the microfluidic-chip (200) is made of a transparent elastomer(PDMS) lined with two main microfluidic chambers (203) (FIG. 18A-1 andFIG. 18A-2) separated by a thin porous membrane 208 (FIG. 2). Thegeometry of the vascular compartment (lower channel in FIG. 18A-1 andFIG. 18A-2) was slightly modified to incorporate an additionalanticoagulant port, which is an element used for eluent sampling anddownstream analysis of soluble biomarkers as described herein. Notably,while in a lung-on-a-chip design endothelial cells were lining only thelower surface of the porous membrane, here we applied a robust protocolto ensure full surface coverage of the vascular compartment andformation of a whole lumen of human endothelial cells. Within 48 hourspost-seeding, endothelial cells formed a confluent monolayer as shown bythe expression of intercellular junctional VE-Cadherin (FIG. 18B)covering the entire surface of the chamber. When perfused with cellculture medium, the endothelial cell monolayer remained stable for atleast 6 days post-seeding, as confirmed via light microscopy and avascular permeability assay (FIG. 19A and FIG. 19B).

The internal surface of the microfluidic system was then coated withType I collagen and fibronectin before seeding human umbilical veinendothelial cells (HUVEC). In some embodiments, collagen is used to coatthe walls of the chip for inducing cell attachment. In some embodimentsa mixture of collagen and fibrin is used to coat the walls of the chipfor inducing cell attachment.

A. Providing a Monolayer of Cells within the Chip Channel.

Cells were seeded in two steps, first onto the bottom surface then ontothe apical surface of the vascular chamber to obtain an evendistribution of cells along the whole microfluidic channel. Within 48hours post-seeding, endothelial cells formed a compact monolayer asshown by the expression of intercellular junctional VE-Cadherin (FIG.18B stained VE-Cadherin), such that a layer of cells are on the internalwalls (not just on the membrane) so that every surface has a cell layercovering the entire surface of the chamber. When perfused with cellculture medium, the endothelial cell monolayer remained stable for atleast 6 days post-seeding, as confirmed via light microscopy (FIG. 19A)and a vascular permeability assay (FIG. 19B).

B. Characterization of Vessel On-Chip with Activating Factors and anAnti-Platelet Drug: Recapitulating Thrombosis On-Chip.

To test the actual thrombotic activity of our biomimetic vessel-on-chipwe perfused freshly isolated human blood through one embodiment of thedisclosed system.

Briefly, human blood was drawn in citrate buffer and used within 4hours. Platelets were stained using fluorescently labeled non-blockingantibodies for the platelet surface marker CD41 and low dosages offluorescent fibrinogen were added in order to visualize fibrindeposition. Blood was re-calcified in order to re-establish the fullcoagulation potential, then introduced in the biomimetic vessel-on-chipfrom the main inlet (embodiments of inlets shown in FIG. 18A-1, FIG.18A-2 and FIG. 19C). The outlet of the chip was connected to a pullingsyringe pump with a system of tubing and connectors made of medicalgrade silicon that excludes any metal components or potential causes ofplatelet activation (FIG. 5A).

After 14 minutes of perfusion, the flow was interrupted and the chipswere immediately imaged via fluorescence microscopy (FIG. 18C and FIG.19D). In healthy conditions, endothelial cells will provide anantithrombotic surface where blood can flow smoothly, and indeed wedetected minimal platelet adhesion or fibrin deposition in ourvessel-on-chip after blood perfusion in control conditions, FIG. 18C,Blood.

1. Vascular Endothelium Controls Blood Clotting in a Microfluidic-ChipDevice.

Vascular tissue can also act as a signaling platform for different bloodcells to be recruited under conditions of tissue inflammation³⁸.Previously³⁵, we used endothelial pre-treatment with Tumor NecrosisFactor-α, to mimic tissue inflammation, inducing expression of factorssuch as tissue factor, von Willebrand factor, and adhesion moleculesassociated with coagulation.

For testing on embodiment of a microfluidic-chip device, called aVessel-On-Chip as described herein, we used endothelial pre-treatmentwith TNF-α (50 ng/ml, 6 hours) to mimic tissue inflammation, and solublecollagen a standard platelet activator (sCollagen, 10 μg/ml), a standardplatelet activator frequently used in vitro and in vivo for othersystems³⁹ to explore how the to explore how the Vessel-On-Chip, asdescribed herein, responded to vascular activation and plateletactivation, respectively.

The inflammation of endothelium by TNF-α was verified by observingincreased expression of anti-ICAM1 in the endothelium compared to thecontrol (for example, see FIG. 20). The use of TNF-α or sCollagentreatments on thrombosis in a Vessel-On-Chip led to more aggressivepatterns of platelet aggregation and fibrin deposition on theendothelium, as demonstrated by increased areas of platelet coverage andfibrin signal intensity (FIG. 18E-1, and FIG. 18E-2). Diverse structuralcharacteristics of blood clots induced by the two experimental stimuliwere captured via scanning electron microscopy (SEM) as colored imagesin FIG. 18D. In particular, TNF-α pre-treatment of the vascularendothelium induced formation of compact clots composed of red bloodcells and platelets, surrounded by fibrin (FIG. 18D, TNF-α.

In contrast, thrombosis by sCollagen involves direct activation of theclassic intrinsic coagulation pathway, which leads to general fibrinformation and parallel activation of platelets by binding of sCollagento their integrin receptor α2β1⁴¹. Blood incubated with sCollagen formeda meshwork of complex fibrin-rich clots that incorporated red bloodcells and platelets FIG. 18D, sCollagen). Additionally, the remarkablealteration of red blood cell morphology (FIG. 18D, sCollagen) isassociated with retraction of fibrin during later stages of bloodclotting^(42,43). The SEM images provide convincing evidence of de novoformation of fibrin rich clots in vitro, a relevant pathophysiologicalendpoint for thrombosis.

These differences are consistent with the mechanism for thrombosis byboth agents, i.e. thrombosis by TNF-α is primarily driven by activationof the endothelium and release of factors that promote adhesion andplatelet-to-platelet interactions which then leads to local thrombinactivation, fibrin formation and clot stabilization⁴⁰.

2. Testing Anti-Coagulation Compounds in A Vessel-On-Chip.

In order to continue a functional characterization of our thrombosismodel, we challenged the two main pro-thrombotic conditions (TNF-α andsCollagen) using Eptifibatide. Eptifibatide refers to a small cyclicheptapeptide capable of blocking platelet aggregation by mimicking theactive residue of fibrin involved in platelet aggregation during bloodclotting, thus inhibiting integrin αIIb/βIII³⁴, the endogenous plateletreceptor for fibrinogen. Eptifibatide was approved by the Food and DrugAdministration (FDA). When used in a Vessel-On-Chip at a clinicallyrelevant concentration of 2 ug/ml⁴⁴ it significantly inhibited plateletaggregation and fibrin clot formation when the endothelium was inflamedwith TNF-α, but its inhibitory effect was modest following treatmentwith sCollagen (FIG. 18E, n=4). Image analysis of multiple experiments(FIG. 18E, n=4) revealed that platelet adhesion was completely inhibitedbut a significant amount of fibrin signal was still detectable insamples treated with TNF-α (FIG. 19D), suggesting that fibrin depositionover an inflamed endothelium might happen independently from plateletaggregation. This finding is consistent with the mechanism of action ofsCollagen, which binds to a different platelet integrin receptor andwhich stimulates coagulation via the platelet-independent intrinsicpathway.

FIGS. 18A1-A22 through FIG. 18E-1 and FIG. 18E-2 demonstrates oneembodiment of On-chip reconstitution of thrombosis showing schematicillustrations of an exemplary embodiment for a microfluidic-chip,micrographs of cells and charts comparing blood clotting events inducedby several compounds. FIG. 18A-1, schematic representation of oneembodiment as a Thrombosis-On-Chip (200). FIG. 18A-2 schematicrepresentation of the chip (200) showing the main features: inlet port(1810), main channel and imaged area (1820), outlet port (1815), and theon-chip anticoagulant port. FIG. 18B Top, endothelial morphology byfluorescent VE-cadherin staining (stain as white lines). Bottom, highmagnification section of endothelial cells stained for VE-cadherin. FIG.18C shows that under control conditions (whole blood alone) plateletsand fibrin shown as white spots and line, are sparse and detectable atthe edge of the imaged areas. Endothelial exposure to TNF-α orpre-incubation of blood with soluble collagen (sCollagen) led toformation of a high number of larger aggregates containing a plateletrich core decorated with fibrin. FIG. 18D shows scanning electronmicrographs of cells showing typical ultrastructure of blood clotsformed on-chip, in control conditions (‘Blood’) the sparse platelets aredispersed on an endothelial surface, in stimulated conditions (‘TNF-α’and ‘sCollagen’) activated platelet aggregates and fibrin networks withtrapped red blood cells are attached to an endothelial cell surface.FIG. 18E-1 and FIG. 18E-2 shows charts showing FIG. 18E-1 plateletcoverage and FIG. 18E-2 fibrin deposition (both charts using the lowertreatment key shown in FIG. 18E-2) that were significantly increased instimulated Platelet coverage, fibrin deposition were significantlyincreased in stimulated, pro-thrombotic conditions in multiple donors(n=4, S.E.M., *p<0.05, ns=not significant), and the effects weresuppressed by adding the drug Eptifibatide. Overall, there is a highlysignificant difference between the TNF-alpha treated with and withoutEptifibatide and between sCollogen treatment with and withoutEptifibatide.

3. On-Chip Anti-Coagulation Allows Sampling of Outflowing Blood:Vessel-On-Chip Biomarker Assessment.

Microfluidic chambers were described as models of thrombosis ((Tsai, etal., In vitro modeling of the microvascular occlusion and thrombosisthat occur in hematologic diseases using microfluidic technology. J.Clin. Invest 122:408-418 (2012); Neeves, et al., The use ofmicrofluidics in hemostasis: clinical diagnostics and biomimetic modelsof vascular injury. Curr. Opin. Hematol. 20:417-423 (2013); Westein, etal., Atherosclerotic geometries exacerbate pathological thrombusformation poststenosis in a von Willebrand factor-dependent manner.Proc. Natl. Acad. Sci. U.S.A. 110:1357-1362 (2013); Westein, et al.,Monitoring in vitro thrombus formation with novel microfluidic devices.Platelets 23:501-509 (2012). including endothelium (Branchford, et al.,Microfluidic technology as an emerging clinical tool to evaluatethrombosis and hemostasis. Thromb. Res. 136:13-19 (2015); Li, et al.,Microfluidic Thrombosis under Multiple Shear Rates and AntiplateletTherapy Doses. PLoS ONE 9:e82493 (2014)) and using imaging as thefunctional readout to study blood clotting. Furthermore, because bloodeventually coagulates inside system components and tubing, eluentsampling from microfluidic chambers becomes virtually impossible. Infact, once blood coagulates in any of these published microfluidicchambers, the cells become virtually inaccessible and sampling ofoutflowing blood typically is not possible. Thus, even though inclusionof biomarkers of coagulation to complement functional imaging readoutsof platelet function is desired, the large amount of uncontrolledcoagulation prevents this type of analysis. In other words, bloodeventually coagulates inside system components and tubing, eluentsampling from the on-chip vessel becomes virtually impossible.

Aiming to overcome these limitations of previous microfluidic devices,we added at least a third microfluidic channel (i.e. an additivechannel) to the outflow port of a chip, forming one embodiment of anOn-Chip device, alternatively a microfluidic chip device, whereanticoagulants, e.g. sodium citrate or EDTA, are introduced throughinput channels and are mixed with blood upon flowing out of themicrofluidic chip (embodiments illustrated in FIG. 12, FIG. 18A-2; FIG.19C; FIG. 22A), for exemplary embodiments of a microfluidic On-Chipdevice. Thus, a microfluidic On-Chip device is contemplated to provide astable platform for assessment of thrombosis.

In order to functionally test the system, re-calcified blood wasperfused through the inlet while anticoagulant, e.g. citrate buffer, wasintroduced online from the port situated next to the outflow port. Thus,blood obtained from the effluent of chips equipped with theanticoagulant port or without anticoagulant port were compared (FIG. 6).From a qualitative point of view the difference was striking.Introduction of sodium citrate through the anticoagulant port allowedfor collection of soluble (not clotted) blood at the end of eachexperiment that remained in the liquid status (FIG. 6).

Thrombin converts fibrinogen into fibrin during clot formation, andanti-thrombin plays a role in maintaining homeostasis by inhibiting theeffect of thrombin. Formation of TAT in the microdevice confirms thatlocal and intrinsic generation of thrombin, a potent platelet agonist,occurs in the model and that counter regulatory mechanisms forcoagulation are retained. Thus, blood sampled from the Vessel-On-Chipwas analyzed for thrombin anti-thrombin complex (TAT), an acceptedclinical biomarker for procoagulation^(48,49). TAT refers to a factorreleased upon activation of the coagulation cascade and one of thebiomarkers associated with thrombotic events occurring in patientsaffected with deep vein thrombosis (DVT) or Systemic lupus erythematosus(SLE)^(45,46). An enzyme-linked immunosorbent assay (ELISA) was used toquantify the thrombin anti-thrombin complex (TAT).

We discovered that levels of TAT (FIG. 22B) were significantly increasedfollowing treatment with TNF-α or combined hu5C8/sCD40L, and minimallyincreased with sCollagen, demonstrating a good correlation with theimaging endpoints described herein. Furthermore, a 3D movie captured theformation of a blood clot induced by IC5c8 treatment as microthrombitrapped within a fibrin meshwork including platelets and nucleate cells(DAPI staining). A still image of an induced clot in the Vessel-On-Chipis shown in FIG. 23.

In addition to anti-thrombin, evidenced by TAT formation, mRNA levels ofthe SERPINE class of inhibitors of blood coagulation proteases,plasminogen activator inhibitor-1 (PAI-1) and SERPINE-2 (Serpin Family EMember 2), were increased 8- and 2-fold, respectively (FIG. 24). Therewere no observed changes in D-dimer in eluates from blood treated withhu5C8/sCD40L combined, suggesting that the rate of procoagulationexceeded fibrinolysis in the assay conditions or that longer incubationtimes may be required to observe formation of fibrinolytic products.

Levels of TAT were increased following endothelial pre-treatment withTNF-α or when blood was activated with sCollagen, showing a goodcorrelation with the imaging endpoints described above.

Surprisingly, Eptifibatide treatment did not inhibit the TAT incrementassociated with INF-α-induced inflammation while it entirely suppressedthe TAT increment due to sCollagen treatment. Apparently, thebiochemical pathway leading from TNF-α-induced vascular inflammation toactivation of the coagulation cascade is independent of plateletaggregation as shown on the disclosed microfluidic chip device (in oneembodiment, as a Thrombosis-On-Chip) which recapitulated the phenomena.

We conclude that the biomimetic Vessel-On-Chip (as a Thrombosis-On-Chip)allows for both qualitative and quantitative assessment of eventscharacterizing blood clotting. The system is indeed able to recapitulateclinically relevant aspects of thrombosis including platelet adhesion,aggregation, fibrin deposition and release of biomarkers ofprocoagulation, such as TAT, in addition to characterizing bloodclotting. Thus, a microfluidic chip device as described herein, providesa unique capability to study real time thrombotic events inmicrophysiological system.

4. In Vivo Thrombosis induced By Hu5C8, a Candidate TherapeuticMonoclonal Antibody, is Mimicked In Vitro by Preformed IC_(5c8).

Once the robustness of the biomimetic Vessel-on-chip was established, inpart as defined endpoints for thrombosis measurements as shown herein,we used the system to study the pro thrombotic effects of theanti-sCD40L monoclonal antibody, hu5c8. Hu5c8 (Ruplizumab) refers to ahumanized monoclonal IgG1 antibody against CD40L alternatively namedanti-sCD40L or anti-CD154. Hu5c8 blocks the interaction of CD40 with itsligand CD154 (CD40L) thus blocking T-cell: B-cell interactions inantibody mediated autoimmune disorders, such as systemic lupuserythematosus (SLE) where it was tested in a clinical trial. However,CD40L is rapidly expressed on the surface of platelets and is releasedin a soluble form after platelet activation and thrombus formation.

Several human clinical trials with the immunosuppressant antibody-baseddrug, hu5C8, were terminated due to unexpected thrombosis andcardiovascular events in patients. These life-threatening side-effectswere not discovered during preclinical testing due to the lack ofpredictive assays. Here we show that a biomimetic vessel on-chip candetect the thrombotic effects of hu5C8. The vessel-on-chip containsmicrocultures of human endothelium and flowing human whole blood, and itrecapitulates complex endpoints for thrombosis, including endothelialactivation, platelet adhesion, platelet aggregation, fibrin clotformation and expression of clinically relevant biomarkers. The dataproduced with our on-chip system is consistent with data from the clinicand other human-relevant tests, highlighting the major significance ofthis on-chip assay for future preclinical evaluation of drug candidates.

Platelet activation assays conducted to study thrombosis risk for thismolecule typically use optimized but not clinically relevantstoichiometric ratios of hu5C8 and se D40L to generate high-orderedimmune complexes (ICs), which is useful for mechanistic studies, butless relevant for assessment of risk for clinical use. Further, based onprevious studies, hu5C8 is able to bind the trimeric form of sCD40L in anon clinically relevant 3 to 1 stoichiometric ratio to form ICs(IC_(5c8)) that ultimately cause rapid platelet activation (illustratedin FIG. 25A)^(12,21)

We investigated whether Hu5c8 added to a biomimetic Vessel-On-Chip wouldbe able to recapitulate the thrombotic events associated with theanti-CD154 mAb hu5C8. We leveraged the physiological realism of aVessel-On-Chip so we tested physiologically relevant concentrations ofan IC_(5c8) preparation made with a ratio of 30,000:1 at clinicallyrelevant doses of hu5C8 (240 μg/ml)⁴⁷, benchmarked to a dose of 20 mg/kgin cynomolgus monkey, to determine whether we could produce a detectablethrombotic effect on the biomimetic vessel. This is the same dose inhumans that caused thrombosis²⁸. We also used disease relevantconcentrations of sCD40L (10 ng/ml), which are typical values reportedin human lupus patients (Kato, et al. “The soluble CD40 ligand sCD154 insystemic lupus erythematosus,” Journal of Clinical Investigation.104(7): 947-955, 1999).

Blood alone or blood treated with hu5C8 alone (240 μg/mL), sCD40L (10mg/ml) alone or with combined hu5C8/sCD40L from 4 donors was processedand perfused through the biomimetic vessel at a flow rate of 60μl/minute, which yields a wall shear stress (0.5 Pa, 5 dyne/cm²)comparable to values found in veins under physiological conditions⁴⁷.

There were no significant treatment-related effects with sCD40L or hu5C8compared to untreated blood, whereas treatment with hu5C8/sCD40Lcombined promoted platelet aggregate formation and fibrin deposition onthe endothelium (FIG. 25B, FIG. 25C). In line with the hypothesis thatbinding of hu5C8 to sCD40L promotes platelet activation and aggregation(FIG. 25A), ultimately causing thrombosis in vivo, scanning electronmicroscopic imaging of the vessel-on-chip perfused with blood containinghu5C8/sCD40L combined revealed the presence of small microthrombi richin fibrin (FIG. 21B). Additionally, image analysis of platelet coverageconducted on 4 different donors all tested in duplicates, confirmed thatthe combination of hu5C8 and sCD40L rather than hu5C8 or sCD40L alone,promotes higher clot formation within the biomimetic vessel-on-chip(FIG. 21D). Modest, but significantly increased expression of vonWillebrand Factor (vWF), Platelet-Endothelial Adhesion Molecule-1(PECAM-1, CD31), and CD40 were observed in samples treated with combinedhu5C8/sCD40L but not other tested samples, suggesting activation of theendothelium (FIG. 21E).

5. C_(5c8) Mediated Thrombosis On-Chip Requires FcγRIIa Interaction.

Mechanistic studies^(26,50) using platelet assays suggest that ahigh-ordered ICs of hu5c8 and sCD40L activate platelets via interactionof IgG with FcγRIIa^(22,50) receptors expressed on platelets. Recentstudies conducted on humanized mice expressing the human Fcγ receptors(FCGR2A) have shown that this receptor plays a role in thrombosisleading to the IC-mediated toxicity^(12,16,21). Notably, the authors ofthe study reported evidence of platelet aggregation and pulmonarythromboembolism within 10 minutes after injecting the mice withpreformed ICs. In order to investigate if the IC_(5c8)-inducedthrombosis that we observed in our vessel-on-chip system also relies onan Fc-mediated mechanism, we used two different tool molecules: IgG2σand IV.3 (FIG. 25A). The IgG2σ variant of hu5C8 has an engineered Feregion⁴⁸ that eliminates affinity for the FcγRIIa, while the monoclonalantibody IV.3 is a blocking antibody binding to FcγRIIa. Briefly,equimolar concentrations of IgG2σ or hu5c8 were incubated with 10 ng/mlof sCD40L for 20 minutes to form IC_(IgG2σ) or IC_(5c8), respectively.

Some of the blood samples were treated with IV.3 (1 μg/ml) for 10minutes to block the FcγRIIa, and then incubated for 20 more minuteswith IC_(5c8). Finally, blood alone (control) or blood incubated withIC_(5c8), IC_(IgG2σ) or IV.3 was perfused through the biomimeticmicrofluidic-on-chip for about 10 minutes. We tested 15 donors and allthe tested conditions described above were tested in duplicates andanalyzed as percentage of platelet coverage based on fluorescencemicroscopic imaging (FIG. 25B). Five of the donors were also tested forfibrin deposition (FIG. 25C). Finally, effluents plasma obtained from atotal of 7 of the donors (including the 5 mentioned before) were used toassess TAT via ELISA (FIG. 25D). Results of platelet coverage and fibrindeposition were normalized with respect to blood alone per each donortested in order to reduce the sample-to-sample variability naturallyoccurring in blood samples. Both the use of IgG2σ variants or FcγRIIablockage via IV.3 suppressed the hu5C8-mediated thrombosis, which isconsistent with results that others obtained with 5c8 with mutatedlow-affinity Fc regions¹³. Notably, there is a large variation indonors, with some responding strongly and others none to the IC_(5c8) atall.

We have observed that some donors have higher sCD40L in the blood and wespeculate that this might be one of the factors contributing thisdonor-to-donor variability (data not shown). The fact that not all blooddonors show a pro-thrombotic effect when treated with IC_(5c8) matchesthe observation that thrombotic and thromboembolic complications ofhu5C8 in clinical trials⁴ were relatively rare. In addition, RNA fromthe chip was analyzed for expression of pro-thrombotic markers includingvWF, CD40 and PAF-1. We found that IC had increased expression ofpro-thrombotic markers (FIG. 24).

As described herein, we have reported and demonstrated a novelbiomimetic, microfluidic system containing an on-chip blood vessel thatcan be used to detect early stages in drug-induced thrombosis andthromboembolism. The system includes at least three elements that arenecessary for studying blood clotting: a confluent endothelial tissue,human whole blood with an active coagulation cascade and physiologicallyrelevant shear forces. We have demonstrated that the system can be usedto analyze multiple aspects of thrombosis, such as platelet adhesion,aggregation, fibrin formation and TAT release, all in a single assay andsome of which in real-time.

Moreover, we have shown that the system mimics pro-thrombotic responsesdue to vascular endothelial activation and sCollagen-mediatedcoagulation and platelet activation. Notably, thrombotic eventsrecapitulated in the system can be inhibited by clinically relevantdosages of the anti-platelet medical drug Eptifibatide.

Additionally, we have shown that the relatively rare and mechanisticallycomplex pro-thrombotic effect of the anti-CD154 mAb hu5C8 can berecapitulated in our system at clinically relevant dosages. Hu5C8mediated increases in platelet adhesion, fibrin clot formation, andincreases in TAT release in the presence of pathophysiologicalconcentrations of sCD40L were all attenuated by blocking interactionswith FcγRIIa or by use of a hu5C8 variant with a low binding affinity toFcγRIIa. These findings provide confidence that there is a low risk forthrombosis in the clinic for a new generation anti-CD154 mAbs that havebeen modified not to interact with FcγRIIa receptors²³. Taken together,our results clearly demonstrate that the micro fluidic-on-chip bloodcould be used as a safety model to de-risk issues related to thrombosisin the drug development process, and potentially as an efficacy modelfor discovery of anti-thrombotic compounds and dissection of complexmolecular mechanisms. The studies described herein, reinforces theconclusions from other studies on hu5C8-mediated thromboembolism. Theresults of our study unequivocally demonstrate the potential added valueof microfluidic-on-chip technology in the preclinical testing of medicaldrugs.

Additional references herein incorporated by reference:

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In some embodiments, the collagen solution for coating of themicrochannels is prevented from entering an “on-chip” reservoir.Prevention includes, but is not limited to, using an amount of coatingsolution that is less than the channel volume. For example, when achannel volume is 4 ul, 3.5 ul of collagen solution is added, in partbecause the entire channel is not coated. In some embodiments, acollagen solution is used for coating 4 and 8 microchannel chips. In oneembodiment, the entire chamber was coated, prior to the addition ofcells, with extracellular matrix (ECM), e.g. fibronectin, variouscollagen types or combinations thereof, including but not limited to anexemplary ECM consisting of a mixture of rat tail collagen I andfibronectin.

V. Exemplary Methods of Using Embodiments of A Microfluidic-Chip Device.

In one embodiment, a blood sample is drawn into a tube containinganticoagulant to inactivate the coagulation cascade at collection. Thesample can be tested or evaluated in a microfluidic device or chip. As aportion of the sample enters the chip (e.g. via an input port), asolution of calcium and magnesium (present in one or more additivechannels positioned at or near the input port in fluidic communicationtherewith) is introduced into that fraction of the sample making contactwith the solution. The reagents in the solution re-activate the nativecoagulation cascade, but only for that portion of the blood samplemaking contact with it. The active blood (e.g. blood capable ofclotting) flows through the chip, e.g. through the microchannel. Wherethe microchannel contains cells, the active blood can interact withthese cells within the “active” region of the microchannel.

In a preferred embodiment, the blood exiting the “active” region makescontact (and is even mixed) with additional anticoagulant (present inone or more additive channels in fluidic communication with themicrochannel and/or output port), so that that portion of the sampleexiting the microfluidic channel (and leaving the chip through an outputport) remains substantially clot-free or unclotted. In this manner, theblood remains in a liquid state after testing (i.e. downstream of the“active” region).

In some embodiment, the additive channel at the outlet can be used toadd other reagents (e.g. for staining), fixatives (e.g. for capturingthe cells and platelets in their state immediately after contact withthe cells in the chip), oil to form blood-containing droplets (e.g. forsequestering blood samples from different time-points in the run, andanalyzing them separately afterwards), etc. The addition of an additivechannel near the outlet allows (is a versatile way) quick treatment ofblood samples as they leave the chip. Such treated blood samples arecontemplated to enable downstream analysis including but not limited tonew types of analysis from the use of the additive channel for treatingblood components as it leaves the chip.

Replicates of the microfluidic-chip device may be made and run inparallel or at different times in order to show consistency in theresults, i.e. the data is good.

It is not intended that the present invention be limited by the type oftesting done on the blood (or other fluid) introduced into themicrofluidic device, One aspect described herein relates to a method ofdetermining cell function. In one embodiment, the method comprises (a)flowing a fluid sample over a surface comprising a monolayer of cells ofa first type thereon; and (b) detecting interaction between cells of asecond type in the fluid sample and the monolayer of cells of the firsttype. The function of the cells of the second type in the fluid samplecan then be determined based on the detected cell interaction. In someembodiments, the monolayer of cells of the first type can compriseendothelial cells, and the cells of the second type in the fluid samplecan comprise blood cells, e.g., platelets (see FIG. 10). In oneembodiment, the endothelial cells are living cells. In anotherembodiment, they are fixed cells (i.e. cells treated with a fixative).Accordingly, another aspect provided herein relates to a method ofdetermining platelet function, which comprises (a) flowing a fluidsample over a surface comprising an endothelial cell monolayer thereon;and (b) detecting interaction between blood cells (e.g., platelets) inthe fluid sample and the fixed endothelial cell monolayer.

VI. “Active” Region of the Microchannel.

In some embodiments, a microfluidic chip unit presents an “activevolume,” “active region” or “active area” in which the desiredconditions and fluid-dynamic regimes for flowing blood are re-created,such that a microchannel contains endothelial cells lining the channelsurface enclosing the “active volume.” Designating an active volume areafor observations and measurements is to avoid measuring backgroundactivation of blood, e.g. when it merely touches the micro-devicesurfaces. Designating an active volume area also allows directinvestigation of the interaction of the blood with an endothelial celllayer, thus eliminating edge effects of the cell layer or themicrofluidic channel. Further, an active volume area identifies the samelocations in parallel for different microchannels.

VII. “On-Chip” and “of Chip” Fluid Reservoirs.

In some embodiments, reservoirs containing blood samples are used withtubing for use in fluidic loading of blood samples onto the chip. Insome embodiments, such tubing is flexible. In some embodiments,reservoirs containing blood samples are loaded using materials andmethods that do not use flexible tubing. Instead blood samples may beloaded into on-chip reservoirs using short rigid connectors, such asleurs, for one example, which are attached to syringes. In someexamples, an off-chip reservoir may be loaded with a blood sample theninserted into a modified on-chip reservoir, wherein such modificationallows for receiving the preloaded off-chip reservoir. Thus, inpreferred embodiments, blood samples are loaded into microfluidic chipreservoirs allowing blood samples to flow into microchannels ofmicrofluidic devices without using tubing attached to inlet ports. Infact, in some embodiments, inlet ports are replaced with fluidreservoirs. Thus, in some embodiments, fluid reservoirs are “on-chip”,wherein in one nonlimiting example, said reservoirs are molded into themicrofluidic device. In some embodiments, fluid reservoirs are“off-chip” reservoirs, wherein in one nonlimiting example, a syringe maybe considered an “off-chip” reservoir. In some embodiments, an“off-chip” reservoir may be inserted into the space created “on-chip”during fabrication of the microfluidic device.

In one embodiment, a microfluidic device has “on-Chip” molded fluidreservoirs for receiving blood samples in amounts larger than able toflow into the microfluidic channels at one time. In one embodiment, an“on-Chip” reservoir refers to an opening that is designed to be moldeddirectly into the chip during chip fabrication for providing amicrofluidic device without the use of tubing for inflowing bloodsamples onto a chip. The use of an “on-chip” reservoir, in part,eliminates material concerns from the tubing (one less material to worryabout), and simply leads to fewer parts to “plumb up” i.e. fluidicallyconnect together. Thus, a blood sample is added to a large reservoiropening within the chip then pushed or pumped through the small openinginto the microfluidic channel.

The design of the on-chip reservoir is contemplated to reduce shearforces on blood components, including but not limited to proteins andcells, flowing into and out of circular tubes/channels fluidicallyconnected to reservoirs. Physics principles, in part relating toHagen-Poiseuille's Law, which basically states that shear stressdecreases to the 4th power with increasing radius. Thus when applied totube diameter and shear stress indicates that a small increase in tubingradius has a significant effect on decreasing shear stress. Thus, shearstress in the reservoir and inlet channel is contemplated as low, i.e.little or no effect on sample components, in part because the dimensionsare large compared to the part of the channel wheremeasurements/analysis is made. In another embodiment, a microfluidicdevice has an “on-Chip” reservoir that is not molded into the chip,referring to a reservoir that is filled with a blood sample prior toinsertion into the large opening molded into the chip. In thisembodiment, a blood sample is loaded into a disc shaped reservoir theninserted into the large opening in the chip. In one embodiment, as thedisc reservoir moves, e.g. pushed, into the large opening in the chip, asmaller covered opening in the disc aligns with the end of themicrochannel which snaps open the disc opening for allowing the bloodsample to flow from the disc into the microchannel. Thus in oneembodiment, after alignment of the openings allows fluidic communicationbetween the blood sample in the disc reservoir, gravity pulls the bloodsample through the microchannel. In one embodiment, after alignment ofthe openings allows fluidic communication between the blood sample inthe disc reservoir and the microchannel, a micropump pushes the bloodsample through the microchannel. In one embodiment, after alignment ofthe openings allows fluidic communication between the blood sample inthe disc reservoir and the microchannel, a vacuum or pump attached tothe outflow port pulls the blood sample from the reservoir through themicrochannel and out the outflow port.

In yet another embodiment, a microfluidic device has an “off-Chip” fluidreservoir referring to a fluid reservoir that is not fabricated as partof the chip. Thus an “off-chip” reservoir contains a blood sample thathas a part, or is capable of connection to a part, that in turn iscapable of insertion into at least a portion of the large opening moldedinto the chip for dispensing a blood sample into the chip. including,but not limited to a syringe as one example of a reservoir. As oneexample of a reservoir, a plastic luer connector attaches a syringereservoir to a chip device, such that at least in part, the largeopening in the device reduces shear on blood flowing in from thereservoir. In some embodiments, each channel is prepped and imaged atonce.

In some embodiments, a large “on-chip” reservoir is designed to be largeenough to reduce the shear on the blood introduced into the microfluidicchannels from the reservoir, in part because excessive shear is known toinduce coagulation.

In some embodiments, the opening between the reservoir and microchannelis designed to be large enough to reduce the shear on the bloodintroduced into the microfluidic channels from the reservoir, in partbecause excessive shear is known to induce coagulation.

One embodiment is shown in a schematic drawing as an exemplarysix-channel chip is shown in FIG. 29. FIG. 29 shows a schematic drawingof an exemplary six-channel device, where, in some embodiments, a largeopening as an “on-chip” fluid reservoir is provided at the end of amicrochannel.

VIII. Geometries of Fluid Channels.

The microfluidic channel can be designed in different geometries, forexample, as shown in illustrations in FIGS. 30A-C. In some embodiments,different microchannel geometries can be grouped together, see FIG. 30A.In yet further embodiments, different geometries are grouped togetherfor viewing in one or more microscope or video camera fields of vision.

In some embodiments, different microchannel geometries are individuallyisolated and connected in various way through their inlet and outlet.For instance, FIG. 29 shows a chip where microchannels are arranged inparallel, but they can also be arrange in series. For instance, fortesting different lengths of microchannels, connecting multiple channelunits of the same type or connecting different types in series iscontemplated for testing coagulation effects of different distances offluid flow on blood-cells flowing in single or multiple geometries.

Many variations of geometries are contemplated for use in channeldesign, construction and use. In fact, nonlimiting examples of aspecific geometry of a microfluidic channel, includes but is not limitedto linear, linear with curves, spiral, discontinuous widths, bifurcatingchannels, etc., to individually test the effect of specific geometry onblood flow through the microfluidic device.

Fluids flowing through these devices are not limited to blood. Indeed,microfluidic systems are designed to perfuse different fluid such asblood, plasma, culture medium, etc. Such that, analysis of events withinthese devices are not limited to fluids. Indeed, interactions arecontemplated for analysis of any particle or groups of particles flowingthrough different types of geometric shapes of channels of themicrofluidic devices, including but not limited to particles found inblood, e.g. blood clotting components, red blood cells, white bloodcells, etc.

Biophysical blood cell-endothelial interactions in regions where abruptchanges in the vascular geometry induce complex local hemodynamicconditions that are relevant to disease pathophysiology.

Further, it is not meant to limit the types of geometries or types offluid dynamics, such that other types of geometry for providing customfluidic dynamics of blood flow are contemplated. In fact, although threeof the four geometries shown each have two additive channels fluidicallyattached to one input port, one of the embodiments shown in FIG. 30A,far right has a bifurcating-channel, thus, the microfluidic channel hasthree ports, two, one on each side of the chip without additivechannels, while the third port has additive channels fluidicallyattached to one input port. In one embodiment, one outlet has twoadditive channels, fluidic ally connected to one input port which isused for analysis of the outflow thus coagulation downstream of thesecond channel, without additive channels, is immaterial. Further,because each additive channel input is contemplated to have its ownpump, the second outlet port would not require a second additive channelpump. However, in some embodiments both outlet ports have additivechannels. In this embodiment, each additive channel input port iscontemplated to be attached to a separate pump. In other embodiments,one pump may be used for multiple additive channel input ports. In yetanother embodiment, a microfluidic device contemplated for use has abifurcating-channel geometry without additive channels at the inlet oroutlet ports. In one embodiment shown, the three geometries on the lefthave one port without additive channels with the port on the other endhaving additive channels and one input for two channels.

FIGS. 30A-C shows schematic drawings of an exemplary 4 channelmicrofluidic device illustrating four exemplary embodiments of presetmicrochannel geometries contemplated for use in recreating specificfluidic dynamics of the blood flow. FIG. 30A shows one embodiment of aschematic top view of a 4 channel chip having four exemplary presetmicrochannel geometries with the same Outflow rate, e.g. having a 100 umOutflow, also shown in FIG. 30B and FIG. 30C (bottom view), FIG. 30Bshows a schematic bottom view diagram of an exemplary 4 channelmicrofluidic device. FIG. 30C shows one embodiment of a schematic 3-Dangular view of a 4 channel microfluidic device contemplated for use asa mold for fabricating chips shown in FIG. 30A and FIG. 30B.

IX. EDTA Outflow

In some embodiments, a fluidically interconnected double port is locatedin the outflow part of the device. This technical feature, called “EDTAoutflow” refers to a round channel that intersect the main channel toprovide a continuous mixing of two different fluids and/or solutions. AnEDTA outflow is contemplated for use in at least two different types ofapplications. In the first type of application, an EDTA outflow is usedfor adding compounds in solution, such as drugs, anti-coagulants, etc.,into the incoming fluid (blood, serum, medium) in a continuously manner.In one embodiment of this application the concentration of the incomingsolution will be established according to the specific use or to thespecific final concentration one desires to achieve. Such that, theconcentrations of compounds in the incoming solutions may be chosen froma range of concentrations. In one embodiment the type of solute will bevaried depending upon the effect under experimentation. In oneembodiment the geometry and the dimensions of the “EDTA outflow” channelwill be varied according to the specific use. In one embodiment specificfinal concentration one desires to achieve). In a second type ofapplication the “EDTA outflow” channel is mainly used to add EDTA (orsimilar chelators) to the blood flowing out of the chip with theultimate goal to avoid coagulation of the blood in the outlet port andallow the collection of blood for analysis. In this second type ofapplication is also contemplated the use of the port to directly addfluorescent dies, antibody or other detection solution to screen theblood. This last application is particularly interesting when the aim isthat to detect molecule with short or very short half-life.

Other blood chelators are contemplated including but not limited to:EDTA, Heparin, Citrate, Dimercaprol (2,3-dimercapto-1-propanol),Ethylenediamine, Phorphine and Herne group. “EDTA outflow” channeldimensions might need to be adjusted according to the specific chelatoror solution added. Note: the “EDTA outflow” channel can be used toprevent blood from coagulate inside the channel during experiments thatrequire direct injection of blood from freshly isolated patient (wecontemplate this application for use in personalized medicine screeningand/or testing). “EDTA outflow” channel is a critical feature of thedevise to take in consideration.

X. Viewing/Imaging Microchannels Using Side-By-Side Imaging Fields.

In some embodiments, the active region of a microfluidic channel mayhave optically transparent viewing areas such that observations ofcomponents inside of the microchannel are visible to an observer viewingthe microfluidic device. Thus, at least half of the upper area of theactive area of the microchannel and the chip material in between theactive area and the observer are constructed of optically clearmaterial. In some embodiments, the entire active area of themicrochannel is made of optically clear material. In some embodiments,the entire microchannel is made of optically clear material. In someembodiments, the entire microfluidic chip device is made of opticallyclear material.

A. Viewing/Imaging Microchannels

In some embodiments, a viewer, or any means of enhanced viewing such aswhen using a microscope, e.g. still microscopy imaging during or at theend of the experiment; images (including but not limited to photographs)and videos of fluidic events imaged by video camera; video microscopy;optofluidic microscopy (OFM) (referring to the use of light-sources torecord projection images of objects flowing above a sensor-array, andutilizing this flow to digitally achieve a spatial resolution beyond thepixel size of the sensors, lens-free optical tomographic microscope,lensfree optical on-chip microscopy, in one embodiment based onpartially coherent on-chip holography, including but not limited toportable telemedicine microscopy, cell-phone based microscopy andfield-portable optical tomographic microscopy, etc., can access (view,observe, or see) are contemplated for use with microfluidic chip devicesdescribed herein. In one embodiment, multiple channels are within therange of motion of the microscope stage. In a preferred embodiment,sizes of active areas of channels match the viewing area of the imagingmeans such that the active areas are capable of being viewed within asingle field-of-view of the viewing/imagining means. In other words, allof the microfluidic channels during a single experiment may be viewed.As one example, events in four channels used simultaneously during anexperiment are capable of being imaged within one field of view. In apreferred embodiment, a single image captures multiple experimentalconditions or replicates.

In some embodiments, combinations of systems are contemplated. As onenonlimiting example, coupling automated imaging and segmentation systemswith microfluidic devices is contemplated to increase through put ofsamples.

B. Side-By-Side Imaging Fields

In some embodiments, side-by side imaging fields are located at orbeneath the active area of the microchannel. It is not meant to limitthe outside dimensions of these imaging fields. Thus, in onenon-limiting example, the outside dimensions is defined as that areathat may be observed by a light microscope within a single field-of-viewof the microscope. In some embodiments, there are subdivisions creatingsmaller areas within the outside dimensions of the imaging field. Insome exemplary embodiments, an imaging field may be referred to as aTile, One exemplary Tile Area is 1350 um by 1350 um which is entirelyvisible viewed with an Olympus Light Microscope, see FIG. 31A. In oneembodiment, a 12 Tile is provided, wherein each Tile Area, or quadrant,is 1350 um by 1350 um, forming a 12 quadrant having 4 tiles across and 3tiles length (down), i.e. a total of 5.4 mm across (4 quadrants) and4.05 mm in length (3 quadrants), see FIG. 31B. However, it is not meantto limit either the size of the Tile or the number of Tiles (quadrants).In some embodiments, each of the Tiles (quadrants) is subdivided intosmaller quadrants. In further embodiments, each of the subdividedquadrants is subdivided into smaller quadrants.

In one embodiment, each microchannel of a microfluidic chip isassociated with a set of quadrants, such that observations in that setof quadrants may be linked to events in that microchannel. In someembodiment, subdivisions may be located so that each microchannel islocated in one set of subdivided quadrants. It is not meant that eachmicrochannel is limited to one Tile. Thus, in some embodiments, there ismore than one Tile associated with each microchannel, such that eachmicrochannel may have more than one Tile associated with the channel,including but not limited to the collection area(s)s for thatmicrochannel.

FIGS. 31A-B show exemplary schematic diagrams of Tile Areas andquadrants representing fields of view, e.g. one embodiment for analyzingevents in microfluidic channels, showing fields of view (FOV) asobserved when viewed through an optical system including but not limitedto an Olympus Light Microscope. FIG. 31A shows an outline representingone field of view (FOV) on a microscope stage, when viewed using a10×ocular, e.g. a 1350 um by 1350 um area, i.e. one Tile Area as viewedwith an Olympus Light Microscope. FIG. 31B shows an exemplaryrepresentation of the total viewing area (e.g. as determined by therange of motion of the stage controls) where the total viewing areaincludes but is not limited to 12 Tile Areas, 12 quadrants, for a totalview area 5.4 mm wide and 4.05 mm in length, wherein each Tile Area orquadrant representing one FOV. In one embodiment, twelve (12) tiles arecontemplated for viewing in under a 30 sec frame rate limit (1 frameevery 30 seconds) for photography, including but not limited tovideophotography, of events occurring within at least one active regionin a microchannel.

Thus, in some embodiments, microfluidic chips described herein, furthercomprise Tile for providing a means, in part, to compare events inidentical locations within each microchannel for making comparisonsbetween samples. In other words, any microfluidic chip described hereinmay further comprise a Tile, e.g. a 12 Tile.

XI. Variations of Microfluidic Devices.

In some embodiments, the microfluidic device are contemplated to have asemi-permeable membrane to separate the fluidic part of the device froma juxtaposed channel or chamber having live mesenchymal and/or epitheliaand parenchymal cells. This setup is desirable when the goal of theinvestigation is to understand the effect of multiple cell types or theeffect on the blood behavior of mesenchymal, epithelial and parenchymalcells in response to external stimuli and stresses.

The cells retained in the membrane region of the device, where cellsthat can communicate through the membrane with the cells seeded into thejuxtaposed channel or chamber.

XIII. Further Embodiments of Additive Channel Designs for Use asMicrofluidic Chip Devices.

Several designs are contemplated for microfluidic Vessel-On-Chips, foruse in analyzing blood clotting. Goal: Design more efficient chip toallow higher throughput testing of various blood conditions. In someembodiments, such chips have endothelial cells, e.g. HUVACs. In someembodiments, such chips do not contain endothelial cells.

In some embodiments, chips may have a single microchannel per sample. Itis not intended to limit one sample to one microchannel per sample.Indeed, in some embodiments, chips may have two or more microchannelsper sample for providing duplicates.

FIGS. 32A-C shows an exemplary schematic diagram of one embodiment of amicrofluidic-chip as 4 total channels on one chip. The four channelshave exemplary dimensions of 100 um diameter channels spaced 50 um apartfor providing an area such that all four channels may be viewed under amicroscope, for one example, within one microscopic field of view.Alternatively, increase channel width to 200 um and test two conditionsat once, for one example, within one microscopic field of view.

FIG. 33 shows an exemplary schematic diagram of features in oneembodiment of a 4 channel chip. The numbers 1, 2, 3 and 4 (numbers nextto dots) represent inlet/outlet ports for attaching to other components,including but not limited to tubing, e.g. for adding or removing fluids,pump(s) or other devices for inducing negative or positive pressure.Each port may be attached to separate or shared channel control(s), asdescribed herein. Multi-inlets (represented by 2 lower dots) are locatedopposite the EDTA inputs (2 upper dots). In one embodiment, multi-inletsrefer to inlets for cell seeding, cell rinsing, e.g. using bufferedsolutions, media, common liquids used in cell culturing, and the like.It is not meant to limit the multi-inlets to hallowing fluids. In someembodiments, Multi-inlets are used for collecting outflowing fluids,e.g. cell media, samples, etc. In one exemplary embodiment, gravitydriven flow for anticoagulants, e.g. EDTA solutions, added into the EDTAinput, provides pressure (gravity) forces for pulsing EDTA into singleadditive channels below, in order to eliminate blood clotting in outflowfrom inlet/outlet ports for collecting and analyzing samples into thereservoirs. Thus in one embodiment, outflow to the reservoirs iscollected from one or more inlet/outlet ports. On chip reservoirs areshown in the lower part of the drawing. As one example for providingpressure forces for pushing sample from the reservoirs through channelsinto the upper portion of the device, height of the reservoirs providessuch pressure. For one example, four reservoirs are shown, eachhaving >70 mm{circumflex over ( )}2 area. In one embodiment, a PDMSdevice height is 15 mm with a volume >1 mL. The lines represent branchesand microchannels, black dots represent locations where 2 branchesmicrochannels merge into one, or where the microchannels connect to areservoir, in one embodiment, one reservoir is connected to each (one)channel.

As one example, FIG. 34 shows an exemplary schematic diagram of oneembodiment of a Thrombosis-On-Chip as 16 total channels on one chip;15.6 mm middle length same as lung-on-chip ˜80 mm wide×50 mm high. Acredit card (left) is shown for a size comparison. The arrow points tomilt-inlet spacing which corresponds to multi pipette spacing. In otherwords, Multi-inlets are spaced 9 mm apart, which is the same spacing as96 well plate where a multi-pipette may be used for inserting samplesand/or solutions. Save significant amount of time with seeding/feedingcells and imaging.

FIG. 34 shows an exemplary schematic diagram of one embodiment of amicrofluidic chip device as 16 total channels on one chip device. Thearrow points to a region containing sample input ports spaced for usewith a multichannel pipetter, i.e. each port corresponds to thedispensing end of the pipette tip, for simultaneously adding samples ineach input port. One exemplary embodiment shows microchannels 15.6 mm inlength. A chip device size comparison is shown at the bottom with aregular sized American credit card on the left compared to the chipdevice outline shown on the right.

A. Exemplary Features for Use in Embodiments of A Microfluidic ChipDevice.

In yet another embodiment of a microfluidic chip device, featuresinclude but are not limited to inlets and outlets for pumps, in part forproviding separate channel fluid flow control, represented at numbersand circles as inlet/outlet ports on FIG. 33; multi-purpose inlets, i.e.multi-inlets, for cell seeding, rinsing, adding and removing commonliquids, such as collagen for coating channels, cell media, celltreatments, e.g. cytokines, etc., represented as circles/ports on FIG.33; EDTA, input ports, for adding anticoagulants, etc., represented ascircles/ports on FIG. 33; and on chip reservoirs, represented as blockedsquares on FIG. 33.

In some embodiments, an EDTA input is located on the upper side of achip such that gravity drives EDTA flow, in part for reducing the numberof pump channels required for use on the entire chip. One (1), 2, 3 and4 corresponds to 4 different devices used in a single experiment. Thus,advantages include but are not limited to sharing EDTA ports betweenconstructs (reducing the need for additional pumps and/or pumpingsystems); joint seeding/washing/reagent ports, since the differentconstructs have to be prepared similarly (for example, seeding channelswith endothelial cells at the same time).

The aim is to reduce the number of EDTA ports and, in turn, thecomplexity of interfacing with and driving many EDTA ports. However,when a single port is used to drive additive channel to severalconstructs, the flow rates in the additive channels may end updifferent. However this difference is undesirable, since a certain EDTAconcentration should be the same in all outputs because a certain EDTAlevel is desired to prevent coagulation. In one embodiment, an EDTAconcentration is 10 uM. In one embodiment, a single EDTA input drives asimilar flowrate into each channel, thus the channel resistance isequal. In one embodiment, the channel lengths are equal. In oneembodiment, resistors may be used for providing a similar, and in someembodiments, an equal flow rate between channels on a device.

Resistors and Regulators: In one embodiment, a device comprises oneinput pressure regulator. In one embodiment, a device comprises two (ormore) input pressure regulators. In one embodiment, a device havingadditive channels for both input (calcium) and output (EDTA) has threeinput regulators. In one embodiment, the flowrate of calcium should be aspecific fraction of the flowrate of input blood and the output. In oneembodiment, the EDTA flowrate is a specific fraction of the output flowrate.

In one embodiment, a pressure regulator pressurizes two reservoirs atonce (e.g. we can divide a current device reservoir into two sections).In one embodiment, one of these reservoirs/sections would be for theinput blood, the other for calcium. In one embodiment, resistors areused in the device, e.g. Pod, for the inlet. In one embodiment, anadditional resistor is used for the calcium. In one embodiment, theresisters provide a single applied pressure to drive flow in twochannels (e.g. blood and calcium), with the ratio of resistances willensure that the two flowrates are at a specific ratio to each other.

For example: a single pressure source on a blood inlet plus a calciuminlet; a single pressure source on calcium inlet plus EDTA inlet and; asingle pressure source on three inlets: blood, calcium, EDTA.

In one embodiment, the use of on-chip reservoirs are contemplated toovercome the material-compatibility of non-anticoagulated blood.

FIG. 33 shows an exemplary schematic diagram of features on oneembodiment of a 4 channel chip. Inlets/outlets for pump/separate channelcontrol are shown as 1, 2, 3 and 4 (numbers and dots). Multi-inletsrefer to inlets for cell seeding/rinsing, common liquids. EDTA inputs.Gravity driven flow for EDTA to eliminate blood clotting for outflowcollection. On chip reservoirs. Four reservoirs, all >70 mm{circumflexover ( )}2 area. When PDMS device height is 15 mm then volume >1 mL.Black lines represent microchannels, showing locations where 2microchannels merge into one, or where the microchannels connect to areservoir, in one embodiment, one reservoir is connected to each (one)channel.

B. Exemplary Methods for Using One Embodiment of a Microfluidic ChipDevice.

An exemplary method of loading and using a microfluidic chip device isprovided for a 4 channel device, such as shown in FIG. 33. To beginloading a device, plug EDTA inputs, and other open ports, such as portslabeled 1, 2, 3, 4 in FIGS. 35A-C and 36A-C.

Thus in one embodiment, unplug EDTA input for providing an outlet foranalysis of outflowing fluid. In one embodiment, pressure fromreservoir, provides pressure force sufficient to push or drive fluidthrough the channels. In one embodiment, height of reservoir, providespressure force sufficient to push or drive fluid through the channels.

In some embodiments, flow is unidirectional. For example, a biologicalsample (e.g. blood or blood components, or other sample) enters themicrofluidic device on one end and proceeds to flow in one direction tothe outlet. In some embodiments, flow is bi-directional. For example,cell seeding (e.g. seeding of endothelial cells) might proceed from aninlet to an active region, growth region, or imaging region; however, abiological sample might proceed in the opposite direction (e.g. bloodfrom an integrated, on-chip reservoir moving into the active region orimaging region from the other side of the chip).

FIGS. 35A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip device related to methods of use. FIG. 35A shows anexemplary schematic diagram of a device during cell seeding, wherepositive pressure, shown by the thick arrows pointing down representingthe direction of fluid flow, is used to seed cells into channels, wherecells are seeded into the multi-inlets while the other ports, 1, 2, 3, 4and EDTA input are plugged (black circles), followed by cell attachmentto the microchannels. Afterwards, medium is pushed through to rinsechannels, see arrowheads in channels/branches between ports and themicrochannels. FIG. 35B shows an exemplary schematic diagram of fluidflow in a device during cell feeding. Medium is added to reservoirs,using 200 ul pipette tips filled with medium inside multi-inlets, whichadditionally serve as plugs during feeding. Pressure used to push mediummay be positive pressure represented by the arrow pointing down, inother embodiments the pressure is negative pressure represented by thearrow pointing up. FIG. 35C shows an exemplary schematic diagram offluid flow in a device during chip prep, where 1, 2, 3, and 4 numberedports are unplugged, while EDTA inlets and multi-inlet ports areplugged. Negative pressure (see direction upwards of thick arrows) isused to fill empty upper channels, then multi-inlets are also plugged.After filling, tubing is attached to inlets 1, 2, 3, and 4 of which atleast one tube is attached to a pump.

FIGS. 36A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip during blood testing. FIG. 36A shows an exemplarydiagram showing where blood is added to reservoirs along with any testagents. Thick arrows show the direction of fluid flow of blood out ofthe reservoirs, with smaller arrowheads showing the direction of flowupwards towards the inlets. FIG. 36B shows an exemplary diagram wherethe four open dots, shown diagonally within the open rectangle (arrow),represent the open (dispensing) ends of pipette tips where the other tipend is attached to a multi-pipetter so that fluid containing an agent,such as a conditional agent, e.g. a coagulation reagent in solution,such as Ca++, intended for adding to blood entering the test channels,is simultaneously added to three ports located below the three lowerdots, one port each for three of the four reservoirs shown as blackareas in the lower part of the chip, where each of the fourmicrochannels is in fluidic communication with a correspondingreservoir. Thus, the solution is mixed into the blood contained in threereservoirs at one time. The remaining reservoir, when receiving asolution as a separate addition into the fourth reservoir port, not inline with the multi-channel pipette tips, upper right, is added/mixedseparately from the other three reservoirs. In some embodiments, thisfourth reservoir is used as a control without the addition of an agentin solution, such as a conditioning solution. FIG. 36C shows anexemplary diagram for preparing Outflow fluid for collection. UnplugEDTA input ports (dots at the top of the diagram), insert the dispensingend of 1 mL syringes for adding EDTA solution. Since a small amount ofEDTA needed, flow downward is gravity driven, see arrowhead pointingdown from the input. Each cm of liquid height=0.1 kPa in pressure; sothat an optimal height of the on-chip device components is calculatedfor each type of chip.

As an example of a method of using a chip, one embodiment of theprotocol might comprise the steps of: plug EDTA inputs, andinlet/outlets 1, 2, 3, and 4. Day 1: Cell growth and maintenance:Seeding: Using positive pressure, a solution containing cells areinserted into, e.g. flowed into a port, for seeding cells into channels;observe cell attachment; then push medium through to rinse channels; andcell feeding: Add medium to reservoirs, leave 200 ul pipette tips filledwith medium inside multi-inlets. Can either use positive or negativepressure. Day 2: Chip testing using microscope observations: unplug 1,2, 3, and 4, then use negative pressure to fill up upper channels; plugmulti-inlets. Attach tubing to inlets 1, 2, 3, and 4, and to a pump. Fortesting a sample, such as blood, add sample with desired agents, such asblood mixed with a test agent, e.g. an anticoagulation antibody. Thenadd a solution for inducing coagulation, such as into ports shown inFIG. 36B.

VIX. Dimensions and Spacing of Fluidic Microchannels for Embodiments ofa Vessel-On-Chip.

It is not intended that the present invention be limited to only certaindimensions for the microfluidic channels. In one embodiment, the widthof a microfluidic channel is 250 um in a microfluidic Vessel-On-Chip. Itis not meant to limit the width of a microfluidic channel. For example,in other embodiments; the width of a microfluidic channel is 400 um. Inyet other embodiments, where more than one channel is present in a chip,the width of microfluidic channels are different, for example, acombination of 250 um channel width and 400 um channel width in amicrofluidic Vessel-On-Chip.

Accordingly, in one exemplary embodiment, the spacing between channelsis 200 um channel spacing for a 4-channel chip. As one non-limitingexample, a 4-channel chip comprises 250 um channel widths with 200 umchannel spacing between channels. As one illustrative example, FIG. 37shows an exemplary schematic diagram of one embodiment of aThrombosis-On-Chip as a 4 Additive Channel Design showing a side by sideviewing area as detailed in FIG. 30B. In one embodiment, a 4 AdditiveChannel Design comprises 4 reservoirs feeding (i.e. in fluidiccommunication) into 1 channel each.

Accordingly, in one exemplary embodiment, the spacing between channelsis 200 um channel spacing for a 8-channel chip. As one non-limitingexample, an 8-channel chip comprises 250 um channel widths with 200 umchannel spacing between channels.

As one example, FIG. 38 shows an exemplary schematic diagram of oneembodiment of a Thrombosis-On-Chip as an 8 Additive Channel Designshowing a side by side tile viewing area.

The arrow on the upper left points to 4 reservoirs that feed (i.e.fluidically connected) into 2 channels, one channel each for aduplicate. The arrow on the middle left points to 8 outlets to collectindividual samples. These 8 outlets may also be used as 8 inlets(circles) for cell seeding/conditional testing (e.g. TNF conditioning).The arrow on the middle left points to a single EDTA inlet feeding 8channels with resistors to equalize flow rate (e.g. separate pumpconnected) contemplated for use in infusion of EDTA. It is not meant tolimit the number of EDTA inlets. Thus, in some embodiments, an 8 channelAdditive Channel design may have two EDTA inlets, each feeding 4channels. It is not meant to limit the EDTA inlet to EDTA, indeed anyanticoagulant in addition to EDTA may find use for adding to the EDTA

The arrow on the right shows a microscopic viewing area as a Tile.

EXPERIMENTAL Example 1 Materials And Methods

This example describes exemplary materials and methods used during thedevelopment of the present inventions.

Microfluidic Chip Manufacturing And Surface Activation: Chip design andfabrication as used herein, were initially modified versions ofpreviously described chips (See, Huh, D. et al. Microfabrication ofhuman organs-on-chips, Nat. Protoc. 8:2135-2157 (2013). In furtherembodiments, chip designs used herein and contemplated chip designs areunlike previously published chips.

In one embodiment, the surface area of the vascular compartment of amicrofluidic chip used herein is greater in comparison to the originallung-on-a-chip design because the original size was too small foroptimal blood component interaction with endothelium. As describedherein, a 1 mm wide and 200 μm tall chamber provided an increase in sizeof the vascular surface area exposed to laminar flow, as compared to anexemplary lung-on-chip. An anticoagulant port, referred to by severalnames, including but not limited to EDTA input, citrate input, etc., wasadded near the outflow port of the vascular chamber. Thus two ports(anticoagulant port and vascular outflow i.e. outflow) were linkedthrough a microfluidic channel (dimension of 250 um wide by 100 umheight) to allow for perfusion of anticoagulant solution during theexperiment. In some embodiments, modifications were made in thecoagulant port dimension. The outlet of the chip was connected to apulling syringe pump with a system of tubing and connectors made ofmedical grade silicon that excludes any metal components or potentialcauses of platelet activation.

Before cell seeding, chips were sterilized by autoclaving followed byfunctionalizing the polydimethylsiloxane (PDMS) surface using oxygenplasma treatment (100 W, 15 sccm, 40 s; PlasmaEtcher PE-100, PlasmaEtch, Reno, Nev.) afterward incubating with 1%(3-aminopropyl)-trimethoxysilane (APTMES; Sigma) in 100% anhydrousalcohol (Sigma) for 20 min at room temperature, flushed twice with 70%ethanol and twice with water before curing overnight at 60° C.

Cell Culture and Microfluidic Chip Preparation: After PDMS surfacefunctionalization and curing, the entire chamber was coated withextracellular matrix (ECM) consisting of a mixture of rat tail collagenI (100 μg/ml in phosphate buffered saline (PBS); BD Biosciences) andfibronectin (30 μg/ml in PBS; BD Biosciences) incubated at 37° C. for 2hours before washing with PBS.

In order to minimize the biological variability of endothelial cells,two fresh vials (passage 1) of Human Umbilical Vein Endothelial Cellsfrom pooled donors (HUVECs, Lonza, Inc., catalog number C2519A, accessed6-5-2017) were thawed at the start of the study. These HUVECs werecultured in Endothelial Growth Medium-2 (EGM™-2 BulletKit™, Lonza, Inc.,catalog number CC-3162, accessed 6-5-2017) and passaged twice beforebeing frozen at passage 3.

At the start of each experiment, two vials of cells were thawed andexpanded for 3 days in EGM-2. Cells were gently detached with 0.05%Trypsin (BD Biosciences, 2-4 minutes incubation at room temperature) and8×10⁶ cells/ml were introduced into the ECM-coated microchannels. Afterincubating for 30 minutes at 37° C., cell attachment to the bottomsurface of the chamber was assessed by, microscopy. Then, a second flaskof HUVECs was trypsinized and used to seed the upper surface of themicrofluidic chamber by introducing the cell suspension, inverting thechip, and incubating at 37° C. for 30 minutes. Each microfluidic chamberwas gently flushed with EGM-2 twice in order to remove unbound cells,then chips were incubated overnight at 37° C. The next day, chips wereconnected to a syringe pump (Chemyx Fusion 200) and perfused with EGM-2for 3 days (30 μL/hr) to provide continuous supply of fresh media. Onday 3, medium was switched to EGM-2 with low serum (1% FBS, no VEGF) topromote cell synchronization overnight, On day 4, chips were used forblood perfusion experiments,

Cell-monolayer integrity: Transmission light microscopy was routinelyused to assess cell-monolayer integrity prior to each experiment. Beforeevery experiment, each one of the endothelialized channels was inspectedvia light microscopy. Samples showing any sign of discontinuity in theendothelium were discarded.Vascular leakage assay as test for tissue integrity: The ability ofcells to form a confluent monolayer was monitored for one week measuringvascular leakage of dextran (ThermoFisher). In order to measure vascularleakage the vascular channel was perfused with cell culture mediumcontaining fluorescent dextran-cascade blue (3 kDa, ThermoFisher). Theapical compartment was perfused with plain medium (without FBS). Atvarious time points, the effluent from the chip was collected, and therelative fluorescence of the top and bottom compartments were measuredusing a standard plate reader. The endothelial cell monolayer wasobserved to remain stable to at least 6 days post-seeding.TNF-alpha Stimulation: In some experiment endothelial cells werestimulate with 50 ng/ml of TNF (Sigma) for 6 hours in order to inducevascular inflammation before blood perfusion.Experiments with Blood: Blood Samples And Endothelium: Citrated humanblood (Research Blood Components, Cambridge, Mass.) was used within 4hours of a blood draw in order to minimize pre-analytical effects onplatelet function. Before every experiment, each one of theendothelialized channels (i.e. microchannels having an endothelial celllayer) was inspected via light microscopy. Samples showing any sign ofdiscontinuity in the endothelium were discarded.Agents Added to Blood: Blood incubated with combined sCD40L/hu5C8, IC or10 ng/ml of soluble collagen (Soluble Calf Skin, Type I-C/N 101562,BIO/DATA, Corporation, Horsham, Pa. USA) was at room temperature for 20minutes.Cloning, Expression and Purification: Generation of IgG2σ was previouslydescribed (Vafa, O. et al. An engineered Fc variant of an IgG eliminatesall immune effector functions via structural perturbations. Methods65:114-126 (2014)). 5c8 human IgG1 and human IgG2-Sigma Fc variants wereproduced in Sino Biological Inc. Gene fragments encoding the 5c8 heavyand light chains were synthesized and cloned into mammalian expressionvectors. DNA was produced and used in transient transfections withHEK293. Antibodies were purified using Protein A affinity chromatographyand formulated in phosphate-buffered saline (DPBS), pH 7.4.Blood Pre-treatment and Perfusion: Formation of IC (preformed IC):Hu5C8, IgG2σ (control) (both provided by Janssen or provided asdescribed herein) For studies with preformed IC, Hu5C8 or IgG2σ as anisotype control were combined with sCD40L (Tonbo Biosciences, San Diego,Calif.) in PBS, in a ratio of 1 molecule of sCD40L to 3000 molecules ofantibody. After 20 minutes incubation at room temperature, the solutionwas diluted in a blood sample to reach the final concentration of 240μg/ml (Hu5C8) and 10 ng/ml (sCD40L).Platelets: Platelets were labeled with human CD41-PE antibody (10 μL/ml,Invitrogen) directly added to the blood and incubated at roomtemperature for 5 minutes.Platelet Aggregation/Activation: As an exemplary thrombosis inducingagent, 15 μg/ml of sCollagen (BIODATA) was used to promote plateletaggregation (Huh, D. et al. A Human Disease Model of DrugToxicity-Induced Pulmonary Edema in a Lung-on-a-Chip Microdevice. Sci.Transl. Med 4, 159ra147-159ra147 (2012).Anticoagulant Drug: As an exemplary standard antiplatelet(anticoagulant) drug, Eptifibatide was used as described herein.Eptifibatide has a well-known mechanism of action, such that a bolus of180 mcg/Kg is frequently used to treat human patients.⁵⁴ We estimatedthat a concentration of 2.4 μM (about 2 μg/ml) is a clinical relevantconcentration. As described herein for experiments includingEptifibatide, 2 μg/ml of Eptifibatide were incubated with blood samplesfor 15 minutes at room temperature before adding sCollagen or beforeflowing through TNF-inflamed biomimetic vessel.Adding Blood to a Vessel-On-Chip And Flow Conditions: A medical gradeplastic reservoir was mounted on the chip, then 800 μl of the bloodsample was pipetted into the reservoir. The chip was perfused with theblood by withdrawing it at a rate of 60 μl/minute using a syringe pump.The citrated blood was re-calcified 2 minutes after the beginning ofeach experiment by adding 100 μl/ml of a solution containing 100 mMcalcium chloride and 75 mM magnesium chloride to the blood to permitcalcium- and magnesium-dependent platelet function and coagulation,Visualizing Fibrin Deposition: When analyzing the formation of fibrin,10 μg/ml of fluorescently labeled fibrinogen (Alexa 488, Invitrogen) wasalso added to the blood. While fluorescent fibrinogen was barelydetected as a diffused fluorescence, fibrin deposition on inflamed cellsand sparse clots was easily distinguished since fibrin formedfilamentous structures produce a signal intensity that is clearlyobserved above a background threshold.Staining of Endothelial Cells: Samples were washed twice in cell culturemedium and then fixed in acetone/methanol 1:1 at −20° C. for 10 minutes.Each sample was then washed three times in PBS and incubated in blockingsolution consisting of PBS and 10% normal donkey serum (Abcam) for onehour. After blocking, samples were washed in PBS twice and thenincubated with anti VE-Cadherin monoclonal antibody (Abcam) diluted1:100 in blocking solution at 4° C. overnight. The next day, sampleswere washed twice in PBS and incubated for 2 hours at room temperaturewith secondary Alexa Fluor 488 donkey anti-rabbit antibody (Abcam)diluted in PBS then washed twice in PBS. Finally Hoechst (Abcam) wasused to stain cell nuclei at room temperature for 5 minutes, thensamples were washed twice in PBS before being imaged.Fluorescence Image analysis: Chips perfused with blood samples wereimmediately imaged via fluorescence microscopy (Olympus IX83, 10×objective UPLFLN10X2, Circa-Flash4.0 CMOS Camera). Each chamber wasinspected along its entire length and 5 images (about 0.25 cm) werecaptured from the central area of the chamber. Image processing andquantification was performed with an automatic macro compiled in Imageand numerical values corresponding to platelet coverage or fibrinflorescence were collected and statistically analyzed in Graphpad PrismV7.Platelet Coverage: A percentage of signal coverage (or Plateletcoverage) was computed from the binary image as the ratio of brightpixels to the total number of pixels in the image. Image processing andquantification was performed with an automatic macro compiled in ImageJin order to ensure unbiased signal measurements. Numerical valuescorresponding to platelet coverage or fibrin fluorescence (signalintensity) were collected and statistically analyzed in Graphpad PrismV7.Scanning Electron Microscopy Sample Pre-treatment and Imaging:Immediately after fluorescence imaging, the chips were fixed in 2.5%glutaraldehyde in 100 mM sodium cacodylate buffer for 2 hours at roomtemperature. Then, the samples were washed and post fixed in 1% osmiumtetroxide for 1 hour at room temperature. After the postfixation, thesamples were dehydrated in series dilutions of ethanol and finallycompletely dried in a Critical point dryer (Tousimus Autosamsri-815).After mounting, the samples were gold sputtered using a Sputter coatingsystem (Hummer 6.2) and imaged by SEM (Jeol 5600LV).

Thrombin-Anti Thrombin (TAT) measurement via ELISA: ThrombinAnti-Thrombin (TAT) levels were analyzed in plasma from blood flowingout of the chip device by removing a sample from the outflow port. ThusTAT levels were evaluated in sample effluents collected from the outflowport of the chip using the Human TAT ELISA Kit (Siemens Healthineers)according to the user manual.

Gene expression analysis: Total RNA was isolated from the chip using RNAMini kit (Fisher Scientific). Two step qPCR was performed usingSuperScript IV Synthesis System (Fisher Scientific) and TaqMan Mastermix (Fisher Scientific) in QuantStudio3 PCR System (Fisher Scientific).Relative expression of gene was calculated using 2^(−ΔΔCt) methods.Statistical Analysis: The data are presented as mean+standard error ofthe mean (s.e.m.). and P values were obtained (via ANOVA) to compare themeans of at least n=3 independent experiments. Data analysis wasperformed using Graphpad Prism V7 (***p<0.0001, **p<0.001, *p<0.05).

Example 2 Endothelial Cells Control Clotting in the Vessel-On-Chip

This example describes exemplary clotting on the chip that is controlledby endothelial cells.

sCollagen treatment of blood activates blood components whileendothelial pre-treatment with TNF-α mimics (simulates) tissueinflammation. Therefore, sCollagen treated blood was added to aVessel-On-Chip containing TNF-alpha pre-treated endothelial cells(endothelium), for simulating inflammation.

More specifically, TNF-α (50 ng/ml) was added to a Vessel-On-Chip forcontact with the endothelium for 6 hours of incubation. Soluble collagen(sCollagen), a standard platelet activator, was used at 10 μg/ml fortreating blood samples, either prior to adding to a Vessel-On-Chip orthrough an additive channel attached to an intake port as the bloodsample is being added to the intake port.

The use of TNF-α or sCollagen treatments on thrombosis in aVessel-On-Chip led to more aggressive patterns of platelet aggregationand fibrin deposition on the endothelium, as demonstrated by increasedareas of platelet coverage and fibrin signal intensity (FIG. 18E, upperand lower charts, respectively). Diverse structural characteristics ofblood clots induced by the two experimental stimuli were captured viascanning electron microscopy (SEM) as colored images in FIG. 18D. Inparticular, TNF-α pre-treatment of the vascular endothelium inducedformation of compact clots composed of red blood cells and platelets,surrounded by fibrin (FIG. 18D, TNF-α.

In contrast, thrombosis by sCollagen involves direct activation of theclassic intrinsic coagulation pathway, which leads to general fibrinformation and parallel activation of platelets by binding of sCollagento their integrin receptor α2β⁴¹. Blood incubated with sCollagen formeda meshwork of complex fibrin-rich clots that incorporated red bloodcells and platelets FIG. 18D, sCollagen). Additionally, the remarkablealteration of red blood cell morphology (FIG. 18D, sCollagen) is knownto be associated with retraction of fibrin during later stages of bloodclotting^(42,43). The SEM images provide convincing evidence of de novoformation of fibrin-rich clots in vitro, a relevant pathophysiologicalendpoint for thrombosis.

These differences are consistent with the mechanism for thrombosis byboth agents, i.e. thrombosis by TNF-α is primarily driven by activationof the endothelium and release of factors that promote adhesion andplatelet-to-platelet interactions which then leads to local thrombinactivation, fibrin formation and clot stabilization⁴⁰.

Example 3 Testing the Use of an Additive Channel in A Vessel-On-Chip forOn-Chip Biomarker Assessment and Treatment with A Candidate Drug

I. Additive Channel.

In order to functionally test the additive channel (i.e. microfluidicchamber(s)) attached to the outflow port of a Vessel-On-Chip,re-calcified blood was perfused through the inlet port while citrate wasintroduced online from a port situated next to the outflow port, flowingthrough the additive channel. Thus, blood obtained from the effluent ofchips equipped with the anticoagulant port (additive channel) or withoutanticoagulant port were compared (FIG. 6). From a qualitative point ofview the difference was striking. Introduction of sodium citrate throughthe anticoagulant port allowed for collection of soluble (not clotted)blood at the end of each experiment that remained in the liquid status(FIG. 6).

II. Vessel-On-Chip Biomarker Assessment.

Blood sampled from the Vessel-On-Chip outflow port was analyzed forthrombin anti-thrombin complex (TAT), a factor released upon activationof the coagulation cascade and one of the biomarkers associated withthrombotic events occurring in patients affected with deep veinthrombosis (DVT) or SLE^(45,46). TAT is an accepted clinical biomarkerfor procoagulation^(48,49). An enzyme-linked immunosorbent assay (ELISA)was used to quantify the thrombin anti-thrombin complex (TAT).

We discovered that levels of TAT (FIG. 22B) were significantly increasedfollowing treatment with TNF-α or hu5C8/sCD40L combined, and minimallyincreased with sCollagen, demonstrating a good correlation with theimaging endpoints described above. Furthermore, a 3D movie captured theformation of a blood clot induced by IC5c8 treatment as microthrombitrapped within a fibrin meshwork including platelets and nucleate cells(DAPI staining). A still image is shown in FIG. 23.

In addition to anti-thrombin, evidenced by TAT formation, mRNA levels ofthe SERPINE class of inhibitors of blood coagulation proteases,plasminogen activator inhibitor-1 (PAI-1) and SERPINE-2, were increased8- and 2-fold, respectively (FIG. 24). There were no observed changes inD-dimer in eluates from blood treated with hu5C8/sCD40L combined,suggesting that the rate of procoagulation exceeded fibrinolysis in theassay conditions or that longer incubation times may be required toobserve formation of fibrinolytic products.

Levels of TAT were increased following endothelial pre-treatment withTNF-α or when blood was activated with sCollagen, showing a goodcorrelation with the imaging endpoints described above.

III. Treatment of Vessel-On-Chip With a Candidate Drug, e.g. AnAnticoagulant Agent.

This example describes exemplary testing of candidate drugs using a drugtreatment with anti-platelet drug, e.g. FDA-approved Eptifibatide in aVessel-On-Chip.

We challenged the two main pro-thrombotic conditions (TNF-α andsCollagen) using Eptifibatide, an anti-platelet drug approved by theFood and Drug Administration (FDA), that mediates its anti-plateleteffect by inhibiting the integrin alphaIIb/alphaIII³⁴, the endogenousplatelet receptor for fibrinogen. Eptifibatide was used at a clinicallyrelevant concentration of 2 ug/ml⁴⁴.

Eptifibatide significantly inhibited platelet aggregation and fibrinclot formation when the endothelium was inflamed with TNF-α. However,its inhibitory effect was comparatively modest following treatment withsCollagen (FIG. 18E, n=4). Surprisingly, Eptifibatide treatment did notinhibit the TAT increment associated with TNF-α-induced inflammationwhile it entirely suppressed the TAT increment due to sCollagentreatment.

Apparently, the biochemical pathway leading from TNF-α-induced vascularinflammation to activation of the coagulation cascade is independent ofplatelet aggregation as shown on the disclosed thrombosis-on-chip whichrecapitulated the phenomena.

We conclude that the biomimetic vessel-on-chip (as a Thrombosis-On-Chip)allows for both qualitative and quantitative assessment of eventscharacterizing blood clotting. The system is indeed able to recapitulateclinically relevant aspects of thrombosis including platelet adhesion,aggregation, fibrin deposition and release of biomarkers ofprocoagulation, such as TAT, in addition to characterizing bloodclotting. Thus, a Vessel-On-Chip provides a very unique capability tostudy real time thrombotic events in microphysiological system.

Example 4 Hu5C8 (Preformed IC) Causes Thrombosis On-Chip

This example describes exemplary testing of candidate therapeuticantibodies.

This example was intended to investigate whether adverse side effectsfound during human clinical trials would be detected in a microfluidicon-chip embodiment as a Vessel-On-Chip under physiological relevantconcentrations of hu5C8 in the presence of sCD40L. As an exemplarytherapeutic antibody, hu5c8 as an immune complex with sCD40L: IC_(5c8),indeed demonstrated potentially adverse side effects in Vessel-On-Chiptesting mimicking adverse side effects that were discovered during hu5c8human clinical trials but not in pre-clinical testing.

We investigated whether Hu5c8 added to a biomimetic Vessel-On-Chip wouldbe able to recapitulate the thrombotic events associated with theanti-CD154 mAb hu5C8. We leveraged the physiological realism of aVessel-On-Chip by testing physiologically relevant concentrations of anIC_(5c8) preparation. Relevant concentrations were made with a ratio of30,000:1 at clinically relevant doses of hu5C8 (240 μg/ml)⁴⁷,benchmarked to a dose of 20 mg/kg in cynomolgus monkey. We used thisrelevant concentration to determine whether we could produce adetectable thrombotic effect on the biomimetic vessel. This is the samedose in humans that caused thrombosis²⁸. We also used disease relevantconcentrations of sCD40L (10 ng/ml), which are typical values reportedin human lupus patients (Kato, et al. “The soluble CD40 ligand sCD154 insystemic lupus erythematosus.” Journal of Clinical Investigation.104(7):947-955, 1999).

To test for a thrombic event, blood was collected from 4 donors, the asblood alone or blood treated with hu5C8 alone (240 μg/mL), sCD40L (10ng/ml) alone or with combined hu5C8/sCD40L was incubated for 20 minutesat room temperature. Each sample was further incubated for 5 minuteswith fluorescent labels for platelets and fibrinogen. As a standardquality control, the control blood was tested for platelet activationusing p-selection expression. We confirmed that platelets in controlblood were not activated but upon activation by ADP, over 90% ofplatelet was positive for p-selectin.

Re-calcified blood samples were then perfused through the biomimeticvessel with untreated endothelium at a flow rate of 60 μl/minute forabout 10 minutes while sodium citrate was re-introduced through theanticoagulant port. This flow rate yields a wall shear stress (0.5 Pa, 5dyne/cm²) comparable to values found in veins under physiologicalconditions⁴⁷.

Immediately after perfusion, the pump was halted and the vessel-on-chipwas imaged by fluorescence microscopy and the blood from theanticoagulant port was collected. There were no significanttreatment-related effects with sCD40L or hu5C8 compared to untreatedblood, whereas treatment with combined hu5C8/sCD40L promoted plateletaggregate formation and fibrin deposition on the endothelium (FIG. 21B,FIG. 21D). In line with the hypothesis that binding of hu5C8 to sCD40Lpromotes platelet activation and aggregation (FIG. 21A), ultimatelycausing thrombosis in vivo, scanning electron microscopic imaging of thevessel-on-chip perfused with blood containing hu5C8/SCD40L combinedrevealed the presence of small microthrombi rich in fibrin (FIG. 21B).Additionally, image analysis of platelet coverage conducted on 4different donors all tested in duplicates, confirmed that thecombination of hu5C8 and sCD40L rather than hu5C8 or sCD40L alone,promotes higher clot formation within the biomimetic vessel-on-chip(FIG. 21C and FIG. 21D). Modest, but significantly increased expressionof von Willebrand Factor (vWF), Platelet-Endothelial Adhesion Molecule-1(PECAM-1, CD31), and CD40 were observed in samples treated with combinedhu5C8/sCD40L but not other tested samples, suggesting activation of theendothelium (FIG. 21E). Surprisingly, the presence of small microthrombidetected in this microfluidic on-chip device was not previously detectedwith other standard methods. Thus, the use of the microfluidic on-chipdevice described herein has a higher sensitivity level for detectingsmall microthrombi than other methods.

We conclude that our microfluidic system, perfused with re-calcifiedhuman blood, is capable of recapitulating hu5C8-mediated thrombosis atphysiologically relevant concentrations of hu5C8 and sCD40L.

Example 5 Hu5C8 Mediated Thrombosis On-Chip Requires FcγRIIa Interaction

This example describes exemplary evaluation of FcγRIIa Interaction inHu5C8 Mediated Thrombosis.

Combined hu5C8/sCD40L was used in experiments conducted in the presenceof the FcγRIIa blocking antibody IV.3 or with a variant of hu5C8 (IgG2σ)designed not to bind FcγRIIa receptors (FIG. 25A).

Blood from several human donors were aliquoted into groups for thefollowing treatments: controls (PBS), sCD40L, combined hu5C8/sCD40L,combined hu5C8 (IgG2_(σ))/sCD40L combined, and combinedhu5C8/sCD40L/IV.3. Each condition was tested with a minimum of 3 donorsto a maximum of 15 donors, and all conditions were tested in duplicatesand analyzed for platelet coverage (FIG. 25B), fibrin deposition (FIG.25C), or increased formation of TAT measured in the eluates (FIG. 25D).Platelet coverage and fibrin deposition following treatment werenormalized and reported as fold-increase over untreated control valuesfor each donor. There were slight increases in platelet coverage in somedonors (about 2-fold) treated with sCD40L, albeit the mean increase wasnot statistically significant compared to controls. This is consistentwith literature describing donor variability in platelet activation fromblood treated with supraphysiological concentrations of sCD4012¹,suggesting that some individuals may have an inherent increased risk forplatelet activation by pathosphysiological concentrations of sCD40L⁵².

Perfusion of the vessel-on-chip with combined hu5C8/sCD40L resulted in astatistically significant increase in platelet aggregation, fibrin dotformation, and increased levels of TAT. There was donor to donorvariability in pro-thrombotic endpoints in the biomimetic vessel, whichis consistent with literature reports of thrombosis induced byanti-CD154 mAbs; the incidence of thromboembolitic events with hu5C8 was2/18 in a lupus nephritis clinical study¹⁰ and a single incident ofthromboembolism with IDEC-131 in the Crohn's disease clinical trialresulted in termination of the molecule. Because individuals in thedisease population with high endogenous sCD40L levels may be prodromalfor thrombosis, the approaches described herein could potentially beapplied to stratify groups of patients receiving therapy to identify andcarefully monitor individuals that may be at risk. Both the conditionsincluding the FcγRIIa-blocking IV.3 antibody and the non-FcγRIIa bindinghu5C8 (IgG2_(σ)) antibody did not show signs of thrombosis in any of theendpoints described herein.

These results confirm previous reports that the main mechanism ofhu5C8-induced thrombosis is dependent on binding of the IgG2 region ofthe mAb to FcγRIIa.

Example 6 Exemplary Features and Methods For Using Microfluidic Chips

This example describes exemplary features, such as additive channels,and methods for using microfluidic chips.

In one embodiment, a blood sample is drawn into a tube containinganticoagulant to inactivate the coagulation cascade at collection. Thesample can be tested or evaluated in a microfluidic device or chip. As aportion of the sample enters the chip (e.g. via an input port), asolution of calcium and magnesium (present in one or more additivechannels positioned at or near the input port in fluidic communicationtherewith) is introduced into that fraction of the sample making contactwith the solution. The reagents in the solution re-activate the nativecoagulation cascade, but only for that portion of the blood samplemaking contact with it. The active blood (e.g. blood capable ofclotting) flows through the chip, e.g. through the microchannel. Wherethe microchannel contains cells, the active blood can interact withthese cells within the “active” region of the microchannel.

In a preferred embodiment, the blood exiting the “active” region makescontact (and is even mixed) with additional anticoagulant (present inone or more additive channels in fluidic communication with themicrochannel and/or output port), so that that portion of the sampleexiting the microfluidic channel (and leaving the chip through an outputport) remains substantially clot-free or unclotted. In this manner, theblood remains in a liquid state after testing (i.e. downstream of the“active” region).

An exemplary method of loading and using a microfluidic chip device isprovided for a 4 channel device, such as shown in FIG. 34. To beginloading a device, plug EDTA inputs, and other open ports, such as portslabeled 1, 2, 3, 4 in FIG. 34.

FIGS. 35A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip related to methods of use. FIG. 35A shows an exemplaryschematic diagram of a device during cell seeding, where positivepressure, shown by the thick arrows pointing down representing thedirection of fluid flow, is used to seed cells into channels, seededinto the multi-inlets while the other ports, 1, 2, 3, 4 and EDTA inputare plugged, followed by cell attachment Afterwards medium is pushedthrough to rinse channels, see arrowheads. FIG. 35B shows exemplaryschematic diagram of fluid flow in a device during cell feeding. Mediumis added to reservoirs, using 200 ul pipette tips filled with mediuminside multi-inlets as plugs. Pressure used may be positive or negativepressure. FIG. 35C shows exemplary schematic diagram of fluid flow in adevice during chip prep, where 1, 2, 3, 4 ports are unplugged, usenegative pressure (see direction upwards of thick arrows) to fill emptyupper channels, then plug multi-inlets. Afterwards, attach tubing toinlets 1, 2, 3, 4 and attach tubes to a pump.

FIGS. 35A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip device related to methods of use. FIG. 35A shows anexemplary schematic diagram of a device during cell seeding, wherepositive pressure, shown by the thick arrows pointing down representingthe direction of fluid flow, is used to seed cells into channels, wherecells are seeded into the multi-inlets while the other ports, 1, 2, 3, 4and EDTA input are plugged (black circles), followed by cell attachmentto the microchannels. Afterwards, medium is pushed through to rinsechannels, see arrowheads in channels/branches between ports and themicrochannels. FIG. 35B shows an exemplary schematic diagram of fluidflow in a device during cell feeding. Medium is added to reservoirs,using 200 ul pipette tips filled with medium inside multi-inlets, whichadditionally serve as plugs during feeding. Pressure used to push mediummay be positive pressure represented by the arrow pointing down, inother embodiments the pressure is negative pressure represented by thearrow pointing up. FIG. 35C shows an exemplary schematic diagram offluid flow in a device during chip prep, where 1, 2, 3, and 4 numberedports are unplugged, while EDTA inlets and multi-inlet ports areplugged. Negative pressure (see direction upwards of thick arrows) isused to fill empty upper channels, then multi-inlets are also plugged.After filling, tubing is attached to inlets 1, 2, 3, and 4 of which atleast one tube is attached to a pump.

FIGS. 36A-C shows exemplary schematic diagrams of one embodiment of amicrofluidic chip during blood testing. FIG. 36A shows an exemplarydiagram showing where blood is added to reservoirs along with any testagents. Thick arrows show the direction of fluid flow of blood out ofthe reservoirs, with smaller arrowheads showing the direction of flowupwards towards the inlets. FIG. 36B shows an exemplary diagram wherethe four open dots, shown diagonally within the open rectangle (arrow),represent the open (dispensing) ends of pipette tips where the other tipend is attached to a multi-pipetter so that fluid containing an agent,such as a conditional agent, e.g. a coagulation reagent in solution,such as Ca++, intended for adding to blood entering the test channels,is simultaneously added to three ports located below the three lowerdots, one port each for three of the four reservoirs shown as blackareas in the lower part of the chip, where each of the fourmicrochannels is in fluidic communication with a correspondingreservoir. Thus, the solution is mixed into the blood contained in threereservoirs at one time. The remaining reservoir, when receiving asolution as a separate addition into the fourth reservoir port, not inline with the multi-channel pipette tips, upper right, is added/mixedseparately from the other three reservoirs. In some embodiments, thisfourth reservoir is used as a control without the addition of an agentin solution, such as a conditioning solution. FIG. 36C shows anexemplary diagram for preparing Outflow fluid for collection. UnplugEDTA input ports (dots at the top of the diagram), insert the dispensingend of 1 mL syringes for

Day 1: Cell growth and maintenance: Seeding: Using positive pressure, asolution containing cells are inserted into, e.g. flowed into a port,for seeding cells into channels; observe cell attachment; then pushmedium through to rinse channels; and cell feeding: Add medium toreservoirs, leave 200 ul pipette tips filled with medium insidemulti-inlets, Can either use positive or negative pressure.

Day 2: Chip testing using microscope observations: unplug 1, 2, 3, and4, then use negative pressure to fill up upper channels; plugmulti-inlets. Attach tubing to inlets 1, 2, 3, and 4, and to a pump. Fortesting a sample, such as blood, add sample with desired agents, such asblood mixed with a test agent, e.g. an anticoagulation antibody. Thenadd a solution for inducing coagulation, such as into ports shown inFIG. 36B.

Example 7 Exemplary Thrombosis Microfluidic Chip Protocol

This example describes an exemplary protocol for thrombosis on-chipwork. The list of materials/reagents is shown in the table below,followed by a detailed protocol.

ECM Coating APTES 1% (Sigma 281778), 10 ul/ml ethanol Rat tail collagenI 100 μg/mL (Corning 354249) For Outflow chip: 0.5 mg/ml of ER1 in 50 mMof ER2 for 20 min under UV, Rat tail collagen I 100 ug/ml (corning354249) CELLS Main Human Umbilical Vein Endothelial Cells at P5/P6Channel (HUVEC, pooled; Lonza C2519A) MEDIA EGM-2 EGM-2 SingleQuot kit(without GA) in 500 mL bottle of EBM-2 (Lonza CC-3162) + 1% P/S CHIPThrombosis 6 parallel 400 um wide, 100 um tall channels, 3.5 mm Chiplarge inlets Reservoirs 5 mL syringes Tubing Tygon E-3603 1/16″ IDtubing for blood experiments Connecters Nylon barbed straight connectorsfor blood experiments (McMaster 5463K36) EXPERIMENTAL REAGENTS BloodFresh human blood in 3.2% citrate vacutainer (Research Blood Components,Cambridge, MA) Ca/Mg 10X calcium/magnesium solution (100 mM calciumsolution chloride/75 mM magnesium chloride) sCD40L Recombinant humansCD40 Ligand (PeproTech 310-02) CD41 CD41 Mouse Anti-Human Ab (doneVIPL3), PE Conjugate (Invitrogen MHCD4104) IV.3 Anti-Human CD32Antibody, Clone IV.3, FITC (Stemcell Technologies 60012F1) FibrinogenFibrinogen from Human Plasma, Conjugated (Life TechnologiesF13191/F35200) TNF-α Tumor Necrosis Factor-α human (Sigma T6674)Chip Coating

-   1. Treat the chip with Plasma with cycle 3 at 100W, 30s (sterilize    chip, leave chip in a dish and close lid so it remains sterile), or-   2. While running the plasma treatment, prepare 1% APTES in 100%    ethanol-   3. After plasma treatment, bring the chip into the hood-   4. Add 10-15 μL of 1% APTES into the channel and aspirate the    residual solution (extra APTES distorts PDMS surface)-   5. Leave at room temperature for 10-20 min-   6. Flush the channel with 100% ethanol (add ethanol to large outlets    and aspirate from inlet)-   7. Aspirate any residual ethanol from the chip-   8. Dry the chip in oven at 60-80° C. for 30 min to 2 h-   9. Flush channels with PBS twice (add PBS to large outlets and    aspirate from inlet)    -   a) if white residue apparent, leave PBS in channels for 5 mins,        then aspirate-   10. Prepare collagen I (100 μg/mL) ECM solution on ice-   11. Use 100 μL of ECM to fill channel and add droplets on inlets-   12. Add wet paper towel around the dish to prevent evaporation-   13. Incubate the chip at 37° C. overnight or at least 2 h-   14. For Outflow chip, flush channels with PBS twice-   15. Prepare collagen I (120 μg/mL) ECM solution on ice-   16. Use 100 μL of ECM to fill channel and add droplets on inlets-   17. Add wet paper towel around the dish to prevent evaporation-   18. Incubate the chip at 4° C. overnight    Or for Outflow chip:-   Step 1. Treat the chip with Plasma cycle #2 (Sterilization cycle)-   Step 2. Flush the chip with 70% ethanol briefly and then wash with    ER2 buffer twice-   Step 3. Prepare ER1 (0.5 mg/ml in ER2) and add 30 ul in to the    channel-   Step 4. Activate under the UV for 20 min-   Step 5. Wash with ER2, 3 times-   Step 6. Wash with DPBS 2 times-   Step 7. Add 30 ul of 100 ug/ml Rat tail collagen per channel and    wrap in parafilm and incubate at 4° C. overnight

Chip Seeding

-   1. Bring the chip to 37° C. incubator and incubate for 1 h minimum,-   2. Flush ECM coated chip with DPBS and then with EGM-2 medium and    incubate at 37° C. for 30 min prior to seeding-   3. Trypsinize HUVEC flasks using 0.05% trypsin-EDTA for 2-3 min    -   a. Thaw 2 vials into 3 T75 flasks and culture for 3 days to seed        3 chips, or use 2 confluent T75 flasks for 3 chips-   4. Spin at 200 g/1000 RPM for 5 min-   5. Resuspend in 200 μL-   6. Count the cells and dilute to 8×10⁶ cells/mL in medium-   7. Add 30W of cells (8×10{circumflex over ( )}6 cells/ml) in to the    channel and flip the chips to seed on the top of the channel.-   8. Incubate for 30 min at 37° C.-   9. To seed bottom of the channel, add 30 ul of cells and incubate    for 30 min at 37° C. without flip-   10. After 30 min, add 200W of media on top of the inlet and outlet    port to cover the port-   11. And incubate at 37° C. overnight for recovery and further    binding in static

All patents, patent applications, and publications identified areexpressly incorporated herein by reference for the purpose of describingand disclosing, for example, the methodologies described in suchpublications that might be used in connection with the presentinvention. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

The invention claimed is:
 1. A method of using a microfluidic device,comprising: a) providing, i) a microfluidic device, comprising: an inputchannel, an output channel, a test channel, wherein said test channelcomprises an input portion in fluidic communication with said inputchannel and an output portion in fluidic communication with said outputchannel, endothelial cells disposed within at least one portion of saidtest channel; and an output additive channel, wherein said outputadditive channel is in fluidic communication with said output portion ofsaid test channel; ii) a biological sample, iii) an anti-coagulationagent, and b) flowing said biological sample at a first flow rate intosaid input channel to produce a first flow stream that flows in adirection from said input portion of said test channel into said outputportion of said test channel and then into said outlet channel ; and c)flowing said agent at a second flow rate into said output additivechannel to produce a second flow stream that merges with said first flowstream and that flows in said output channel in a direction away fromsaid output portion of said test channel and away from said testchannel, such that said agent contacts at least a portion of saidbiological sample, wherein steps b) and c) can be performed in any orderor simultaneously, and said second flow rate is a fraction of said firstflow rate.
 2. The method of claim 1, wherein said flowing in step b)allows for a thrombotic process.
 3. The method of claim 2, furthercomprising d) optically observing said thrombotic process.
 4. The methodof claim 1, wherein said biological sample comprises blood.
 5. Themethod of claim 1, wherein said biological sample comprises at least oneblood component.
 6. The method of claim 1, wherein said agent isselected from the group consisting of EDTA, citrate, ACD, heparin andcoumarin.
 7. The method of claim 3, wherein said optically observingcomprises live-cell imaging.
 8. The method of claim 7, wherein saidoptically observing comprises live-cell imaging during said flowing ofsaid biological sample.
 9. The method of claim 1, further comprising astep of fixing said cells after step c).
 10. The method of claim 1,further comprising a step of fixing said cells before step d).
 11. Themethod of claim 1, wherein at least a portion of said biological sampleflows out said output channel.
 12. The method of claim 11, furthercomprising the step of collecting at least a portion of said sample fromthe output channel.
 13. The method of claim 12, further comprising thestep of analyzing said sample collected from said output channel. 14.The method of claim 13, wherein said analyzing comprises testing for theexistence of, or the amount of, components in said sample collected fromsaid output channel.
 15. The method of claim 14, wherein said componentsare selected from the group consisting of cytokines, antibodies, bloodcells, cell surface markers, proteins, RNA, DNA, biomarkers and clottingfactors.
 16. The method of claim 1, wherein said device furthercomprises at least one input additive channel.
 17. The method of claim1, further comprising adding a test compound to the agent before stepc).
 18. The method of claim 1, further comprising adding a test compoundto the biological sample before or during step b).
 19. The method ofclaim 1, wherein said microfluidic device further comprises a porousmembrane and a back channel, wherein said membrane is situated betweenat least one portion of said test channel and at least one portion ofsaid back channel.
 20. The method of claim 19, wherein at least onenon-endothelial cell type is disposed within at least one portion ofsaid back channel.
 21. The method of claim 20, further comprisinganalyzing at least some of said cells of at least one non-endothelialcell type after step c).
 22. The method of claim 19, further comprisingd) flowing a third fluid into said back channel so as to create anoutflow of said back channel.
 23. The method of claim 22, furthercomprising analyzing the outflow of said back channel.
 24. The method ofclaim 1, wherein said output additive channel lacks cells.